Impact of hypercapnia on alveolar Na+

Transcription

Institut für Tierphysiologie
Universitäten Gießen und
AG Molekulare Zellphysiologie
Marburg Lungen Zentrum
Prof. Dr. W. Clauss
Prof. Dr. W. Seeger
AG Vadasz
Impact of hypercapnia on alveolar Na+-transport
Establishing a system for ENaC-protein detection
Inaugural Dissertation
zur Erlangung des Doktorgrades Dr. rer. nat.
der naturwissenschaftlichen Fachbereiche
der Justus-Liebig-Universität Gießen
Vorgelegt von Benno Andreas Buchbinder
Gießen, Oktober 2013
Dekan:
Prof. Dr. Volker Wissemann
Interner Betreuer:
Prof. Dr. Wolfgang Clauss
Externer Betreuer:
Prof. Dr. Werner Seeger
Content
Table of Contents
1.
Introduction ......................................................................................................................1
1.1.
Acute Respiratory Distress Syndrome (ARDS) .............................................................1
1.1.1.
Ventilation during ARDS .........................................................................................2
1.1.2.
Permissive hypercapnia ............................................................................................3
1.1.3.
Pathophysiology of ARDS .......................................................................................4
1.1.4.
Alveolar epithelial barrier – function and repair ........................................................5
1.2.
Deg/ENaC superfamily.................................................................................................8
1.2.1.
Common structure ....................................................................................................8
1.2.2.
Various functions .....................................................................................................8
1.2.3.
Epithelial Na+-channels ............................................................................................9
1.2.4.
Regulation.............................................................................................................. 10
1.2.5.
Signaling pathways ................................................................................................ 11
1.3.
2.
State of the art ............................................................................................................ 13
Methods ......................................................................................................................... 15
2.1.
Cell culture................................................................................................................. 15
2.1.1.
Culture of A549 cells ............................................................................................. 15
2.1.2.
Transfection of A549 cells...................................................................................... 15
2.1.3.
Culture of H441 cells ............................................................................................. 16
2.1.4.
Optimization of H441 cell transfection ................................................................... 16
2.1.5.
Transfection of H441 cells...................................................................................... 17
2.1.6.
Isolation of primary rat alveolar epithelial cells ...................................................... 17
2.1.7.
Establishment of ATII cell transfection................................................................... 17
2.1.8.
Final protocol for the Nucleofection of ATII cells................................................... 18
2.1.9.
Flow cytometry ...................................................................................................... 18
2.1.10.
Fluorescence microscopy ....................................................................................... 19
2.1.11.
Cytotoxicity assay .................................................................................................. 19
2.2.
Ussing chamber measurements ................................................................................... 19
2.3.
Real-time rtPCR ......................................................................................................... 21
2.4.
Generation of genetically modified ENaC-constructs .................................................. 22
2.4.1.
Subunit-specific cloning procedure ......................................................................... 24
2.4.1.1.
β-ENaC .................................................................................................................. 24
2.4.1.2.
α-ENaC .................................................................................................................. 25
2.4.1.3.
γ-ENaC .................................................................................................................. 27
2.4.1.4.
Site directed mutagenesis α-ENaC .......................................................................... 28
I
Content
2.4.2.
Transformation....................................................................................................... 29
2.4.3.
Plasmid preparation ................................................................................................ 29
2.5.
Western-Immuno-Blotting .......................................................................................... 31
2.6.
Biotin-Streptavidin-Pulldown ..................................................................................... 32
2.7.
Pulse-chase-experiments............................................................................................. 33
2.8.
Immunoprecipitation of detergent insoluble fraction of ENaC ..................................... 33
2.9.
Statistical analysis and graphical illustration ............................................................... 34
2.10.
Materials .................................................................................................................... 35
3.
Results ........................................................................................................................... 41
3.1.
Functional Ussing chamber measurements .................................................................. 41
3.1.1.
pH changes of the buffer have no influence on alveolar Na+-transport ..................... 41
3.1.2.
Hypercapnia reduces total INa ................................................................................. 43
3.1.3.
Membrane-permeabilization experiments ............................................................... 44
3.1.4.
Long-term hypercapnia affects the apical Na+-conductance .................................... 46
3.2.
rtPCR: Transcription levels of ENaC during hypercapnia............................................ 48
3.3.
Expression cloning of modified human ENaC constructs ............................................ 50
3.3.1.
β-ENaC .................................................................................................................. 50
3.3.2.
Cell surface abundance of β-ENaC-V5 ................................................................... 51
3.3.3.
Expression cloning of human α- and γ-ENaC.......................................................... 53
3.3.1.
Transfection of H441 cells...................................................................................... 59
3.3.2.
Transfection of primary rat alveolar epithelial cells................................................. 64
3.3.3.
ENaC transfection in ATII cells.............................................................................. 69
4.
Discussion ...................................................................................................................... 70
4.1.
Hypercapnia modulated regulation of alveolar Na+- transport...................................... 70
4.2.
Molecular mechanism of CO2-regulated ENaC function ............................................. 73
4.3.
Transfection of H441 cells – Seeking the right system ................................................ 75
4.4.
Novel technique for transfection of primary alveolar epithelial cells ........................... 77
5.
Summary ........................................................................................................................ 79
6.
Zusammenfassung .......................................................................................................... 81
7.
Literature........................................................................................................................ 83
8.
Eidesstattliche Versicherung ........................................................................................... 96
9.
Danksagung.................................................................................................................... 97
10. Curriculum vitae ............................................................................................................. 98
II
Abbreviations
Table 1 Abbreviations
[m/v]
mass per volume
[v/v]
volume per volume
7-AAD
7-Aminoactinomycin
A549
human adenocarcinoma cell line
A6 cells
Xenopus laevis kidney cell line
AD/DA
analog-digital / digital-analog
AECC
American-European Consensus Conference on ARDS
AMPK
adenosine-monophosphate-activated kinase
AQP5
aquaporin 5
ARDS
acute respiratory distress syndrome
ATI / ATII
alveolar epithelial cell type I and II
CaMKK-β
Ca2+-calmodulin dependent kinase kinase β
CD90
cluster of differentiation 90
cDNA
complimentary deoxyribonucleic acid
CFTR
cystic fibrosis transmembrane conductance regulator
CHO
chinese hamster ovary cell line
COS-7
fibroblast-like cell line derived from monkey kidney tissue
Deg
Degenerin
DNA
deoxyribonucleic acid
E-cadherin
epithelial cadherin
Abbreviations
ECL
enhanced chemiluminescence
EDTA
ethylenediaminetetraacetic acid
ENaC
epithelial Na+- channels
ER
endoplasmic reticulum
ERK
extracellular signal-regulated kinase
et al.
et alii
FACS
fluorescence-activated cell sorting
FBS
fetal bovine serum
FIO2
fracture of inspired oxygen
GFP
green fluorescent protein
GRE
glucocorticoid responsive element
h
hour
H441 cells
human lung adenocarcinoma epithelial cell line
HA
heme aggluttinin
HEK-293
human embryonic kidney cell line 293
HPRT
hypoxanthin-phosphoribosyl-transferase
HSC
highly selective channels
IAmi
amiloride-sensitive current
IFN-γ
interferon-gamma
IgG
immunoglobulin G
IKKβ
inhibitor of nuclear factor kappa-B kinase subunit β
Abbreviations
IL-1β
interleukin-1β
INa
electrical current produced by transepithelial Na+-transport
Isc
electrical short circuit current
IU
international unit
JNK
c-Jun N-terminal kinase
kDa
kilo Dalton
LB
Luria broth
LDH
lactate dehydrogenase
LPS
lipopolysaccharide
LSC
low selective channel
MAPK
mitogen-activated protein kinase
MDCK
Madin-Darby canine kidney type 1
MEC
mechanosensory abnormality protein
MG-132
proteasome inhibitor
miRNA
micro ribonucleic acid
mmHg
mm of mercury
mRIPA
modified radio-immunoprecipitation assay buffer
mRNA
messenger ribonucleic acid
MSC
mesenchymal stem cell
N
number of …
n.s.
not significant
Abbreviations
NCBI
National Center for Biotechnology Information
Nedd4-2
neural precursor cell expressed developmentally down-regulated
protein 4-2
NET
neutrophil extracellular traps
NIH
National Institutes of Health
NKCC
Na+,K+,2Cl- cotransporter
P
probability
P0
open probability
P38
p38-mitogenactivated proteinkinase
Pa
partial pressure
PBS
phosphate buffered saline
PEEP
positive end-expiratory pressure
PKC-ξ
protein kinase-C-ξ
PY-motifs
conserved proline rich sequence in all ENaC subunits
RNA
ribonucleic acid
rpm
rounds per minute
rtPCR
reverse transcription polymerase chain reaction
SDS
sodium dodecyl sulphate
SEM
standard error of the mean
SGK1
serum- and glucocorticoid regulated kinase
SNP
single nucleotide polymorphisms
SOC-medium
salt-optimized with carbon (glucose)-medium
Abbreviations
SP
surfactant protein
TAE
Tris-acetate-EDTA-buffer
TBS-T
Tris buffered saline incl. tween 20
TE-buffer
Tris-EDTA-buffer
TNF-α
tumor necrosis factor- α
U
unit
Ub
ubiquitin
UNC
uncoordinated (protein)
V5
epitope tag derived from paramyxovirus of simian virus 5
VE-cadherin
vascular endothelial cadherin
VEGF
vascular endothelial growth factor
VILI
ventilator-induced lung injury
VT
tidal volume
WW-domain
tryptophane rich sequence of a protein, here Nedd4-2
YFP
yellow fluorescent protein
ZO-1
zona-occludens protein-1
Introduction
1. Introduction
1.1. Acute Respiratory Distress Syndrome (ARDS)
ARDS has first been described in 1967, as a life-threatening respiratory condition
characterized by acute onset of tachypnea, hypoxemia and loss of compliance, induced
by different stimuli, including severe trauma, viral infection and pancreatitis (Ashbaugh
et al., 1967). Approximately 190,000 cases and 74,000 deaths each year (mortality ~ 40
%) are estimated for the United States alone, underlining the clinical importance of
ARDS (Rubenfeld and Herridge, 2007).
Until now, intensive research has been done on the mechanism and treatment of ARDS.
The initial definition was revised in 1994 by the American-European consensus
conference on ARDS (AECC) (Bernard et al., 1994) and further modified in the “Berlin
Definition” in 2013 to match clinical criteria and to improve diagnosis of ARDS. The
current definition of ARDS includes the acute onset within one week of a known
clinical insult or new / worsening symptoms, non-cardiogenic bilateral infiltrates and
hypoxemia, with the severity based on the degree of hypoxemia calculated as the ratio
of oxygenation (PaO2) to the fraction of inspired O2 (FIO2) (mild ARDS: 200 mmHg <
PaO2 / FIO2 ≤ 300 mmHg; moderate ARDS: 100 mmHg < PaO2 / FIO2 ≤ 200 mmHg;
severe ARDS PaO2 / FIO2 ≤ 100 mmHg). Since the oxygenation is dependent on the
ventilation of patients, these criteria have to be applied when the patient is ventilated
with a positive end-expiratory pressure (PEEP) higher than 5 cmH2O.
The main causes of ARDS are categorized into direct and indirect. Direct risk factors
include all conditions that directly target the lung, such as pneumonia, inhalational
injury or near-drowning, whereas indirect risk factors can be quite diverse. Major
trauma, non-pulmonary sepsis, severe burns or drug overdose can all trigger ARDS
(Ranieri et al., 2012).
1
Introduction
1.1.1. Ventilation during ARDS
Currently, there are only supportive treatment options for the different kinds of ARDS.
These include the mode of ventilation, conservative fluid management and prone
positioning (Guérin et al., 2013; Wiedemann et al., 2006; Young et al., 2013). PEEP
was recognized to be effective in reducing mortality in ARDS patients already in 1967,
while only a small subset of patients improved with steroids, antibiotics or digitalis
(Ashbaugh et al., 1967). Especially in combination with low tidal volume ventilation it
is highly beneficial, as it prevents the alveoli from collapsing thus keeping the lung
open (Hickling et al., 1994). Subsequently, oxygenation is improved, even more when
combined with fluid management (Wiedemann et al., 2006). But mechanical ventilation
is not only beneficial for the lung. When inadequate pressures are applied, the lung can
be damaged, a condition called ventilator-induced lung injury (VILI) (Biehl et al.,
2013), leading to increased morbidity and mortality (Parsons et al., 2005; Ranieri, 1999;
Ranieri et al., 2000). Optimal ventilation strategies are still under debate. Studies aiming
to elucidate the effect of lower versus higher PEEP are controversial and a metaanalysis in 2010 came to the conclusion that only patients suffering from severe ARDS
benefit from higher PEEP levels (Briel et al., 2010). The authors who published the
most recent meta-analysis conclude that high PEEP levels neither reduced mortality
before hospital discharge, nor significantly increase the risk of barotrauma but improve
oxygenation within the first seven days of treatment (Santa Cruz et al., 2013). But not
only the level of PEEP should be considered when determining the optimal ventilation
settings. Another important variable in ventilating patients is the tidal volume (VT). Low
VT ventilation has been shown to be safe compared to conventional ventilation (6 vs. 12
ml/kg predicted body weight) (Cheng et al., 2005), minimize damage to the lung, reduce
mortality and increase the number of days without ventilator (The Acute Respiratory
Distress Syndrome Network, 2000) and according to a recent clinical trial it also acts
protective on the cardiovascular system (Natalini et al., 2013).
2
Introduction
1.1.2. Permissive hypercapnia
Lung-protective ventilation with low tidal volume and reduced minute-ventilation
(volume of air inspired per minute) leads to a decreased elimination of CO2 from the
alveolar space, ultimately resulting in systemic hypercapnia with or without acidosis.
Under normal conditions humans exhibit a partial pressure of arterial CO2 (PaCO2) of
35-45 mmHg, but during several pulmonary diseases it can even exceed 200 mm Hg
(Connors et al., 1996; Feihl and Perret, 1994; Mutlu et al., 2002; Sheikh et al., 2011).
Clinical studies suggest that slightly elevated CO2 levels are not detrimental for the
patients and can be accepted (Hickling et al., 1994; The Acute Respiratory Distress
Syndrome Network, 2000). Now, permissive hypercapnia as a consequence of lung
protective ventilation is widely accepted, although not without reservation (Curley et
al., 2011). Several clinical studies not only show hypercapnia is not harmful, but they
even support a beneficial effect on the lung and on survival of patients (Ryu et al.,
2012). In a rat model of systemic sepsis induced lung injury for example, hypercapnia
only reduced the severity of lung injury when accompanied by acidosis, while buffered
hypercapnia failed to provide any benefit (Higgins et al., 2009). This is interesting,
because in another infection-based lung injury model sustained hypercapnia worsened
lung injury (O’Croinin et al., 2008). Generally, hypercapnia impairs lung host defense
in different ways. It has been shown to alter the innate immune response (Sporn et al.,
2011), but also phagocytosis (Wang et al., 2010) and generation of reactive oxygen
species (Gates et al., 2011).
pH independent effects of hypercapnia
Positive and negative effects of hypercapnia in vivo cannot be well investigated without
any contribution of pH. But in several in vitro models pH-independent hypercapnia
induced mechanisms have been identified that are highly detrimental in the context of
ARDS. Hypercapnia was shown to inhibit epithelial cell wound repair and the inhibition
was still present, when acidosis was buffered to normal pH (O’Toole et al., 2009).
Highly relevant is the finding, that alveolar epithelial function is significantly impaired
during hypercapnia. This process has also been proven to be pH independent and is
discussed in detail later (Briva et al., 2007; Chen et al., 2008; Vadász et al., 2008;
Welch et al., 2010). Another event that has been demonstrated to be pH independent is
blunting of the immunological response (Helenius et al., 2009).
3
Introduction
1.1.3. Pathophysiology of ARDS
ARDS is characterized by dysregulated inflammation, inappropriate accumulation and
activity of leukocytes and platelets, uncontrolled activation of coagulation and disrupted
epi- as well as endothelial barrier function (Matthay et al., 2012).
Inflammation by activation of the innate immune response is triggered by microbial
products or cell injury-associated endogenous molecules that are recognized by patternrecognition receptors located on the surface of the lung epithelium and alveolar
macrophages (Opitz et al., 2010). Dependent on the cause of the disease, several other
mechanisms can contribute to the inflammatory process, such as formation of
neutrophil extracellular traps (NET) (Caudrillier et al., 2012) or interaction of
inflammatory and hemostatic cells (Looney et al., 2009). Since ARDS can be induced
by many different stimuli, inflammatory processes are not good or bad per se, but need
fine adjustment to clear pathogens from the lung without worsening the damage.
Another important characteristic of ARDS is the disruption of the microvascular and
epithelial barrier causing the accumulation of protein-rich edema fluid, as well as
leukocytes and erythrocytes in the alveolar space thereby impairing gas exchange.
Disruption of the endothelial barrier is caused by destabilization of its major component
vascular-endothelial cadherin (VE-cadherin) by several factors like cytokines (TNF-α,
and IFN-γ), lipopolysaccharide (LPS) (Herwig et al., 2013) or VEGF (Chen et al., 2012)
that are associated with different models of ARDS. Not only increased permeability of
the endothelium is a problem in ARDS. Clinical data link obstruction and destruction of
the microvascular bed in the lung, assessed by the pulmonary dead-space fraction, to
increased mortality of ARDS patients (Nuckton et al., 2002). When only the endothelial
barrier is disrupted, with the epithelium still intact, no increased permeability was
observed and the alveolar epithelial liquid clearance was normal, indicating that a loss
of endothelial barrier function does not necessarily induce ARDS (Wiener-Kronish et
al., 1991).
Less is known about the mechanism of epithelial barrier damage. In patients that died
from ARDS a variety of events are reported to happen at the alveolar epithelium,
including cytoplasmic swelling, vacuolization and necrosis leading ultimately to a loss
of epithelial cells (Bachofen and Weibel, 1977). Taken into account that the epithelial
barrier is much tighter than the endothelial barrier (Taylor and Gaar, 1970) its
disruption is an important event in the development of pulmonary edema. Besides
4
Introduction
prevention of flooding of the airspace another critical function of the alveolar
epithelium is the precise regulation of fluid transport.
Maximal fluid clearance has only been observed in 13 % of ARDS patients which is
highly relevant when considering, that the hospital mortality was 62 % when fluid
clearance was impaired or submaximal, compared to a mortality of “only” 20 % in
patients having maximal fluid clearance rates (Ware and Matthay, 2001).
The resolution of ARDS is quite complex and different events have to be synchronized.
The epithelial and the endothelial barriers have to be reestablished and edema fluid, as
well as inflammatory cells and exudate, need to be cleared from the airspace.
1.1.4. Alveolar epithelial barrier – function and repair
The alveolar epithelium is comprised of two different cell types: Alveolar epithelial
cells type I (ATI) and type II (ATII). ATI cells cover approximately 93 % of the
alveolar surface due to their large, flat morphology, thus keeping the diffusion barrier
thin to enable proper gas exchange (Wang and Hubmayr, 2011). ATII cells are
cuboidal, twice as abundant as type I cells and cover approximately 7 % of the alveolar
surface. They account for 16 % of the all alveolar cells and have half of the volume of
ATI cells (Crapo et al., 1982). Main functions are absorbance of excess alveolar fluid, a
process later described in detail and secretion of surfactant (Kawada et al., 1990; Press
et al., 1982). Surfactant, short for surface active agent consists of different components
(10 % proteins, 90 % lipids) some of which reduce surface tension (surfactant proteins
B and C (SP-B, SP-C) and phospholipids). Others play a role in host defence (SP-A and
SP-D), underlining the immunological importance of ATII cells (Günther et al., 2001).
Unidirectional amino acid and protein uptake from the apical side has also been
described (Buchäckert et al., 2012; Uchiyama et al., 2008).
In the normal lung, proliferation of this cell-type is minimal, with a reported
proliferative subpopulation of 0.5-1 % of all ATII cells (Kalina et al., 1993). In different
models of lung injury in rats ATII cells have been reported to function as progenitors
for type I cells, indicating their importance in reestablishing the alveolocapillary barrier
following lung injury by proliferation and differentiation (Adamson and Bowden, 1974;
Clegg et al., 2005; Evans et al., 1973). In the mouse model of hyperoxia-induced lung
injury ATII cells have been identified to promote alveolar epithelial regeneration
(Adamson and Bowden, 1974). This process seems to be activated generally upon
injury of the epithelium, independent of the specific stimulus.
5
Introduction
Lately, evidence for pluripotent stem cells that differentiate into alveolar epithelial cells
is rising. Those cells have been found in the adult human lung and express markers of
mesenchymal stem cells (MSCs) like CD90 as well as pro-surfactant protein-C that is
associated with ATII cells but can also express aquaporin 5 (AQP5), a marker of ATI
cells. Still, it is not known whether this cell type directly differentiates into ATI or ATII
cells or both (Chapman et al., 2011; Fujino et al., 2011).
Repopulation of the injured epithelium and restoration of the epithelial barrier function
alone is not sufficient to resolve ARDS. Once the epithelium is tight again, the excess
fluid needs to be cleared from the airspace.
Under normal conditions the alveolar surface liquid volume is precisely regulated. By
low temperature scanning electron microscopy of the rat lung an area-weighted average
thickness of 0.2 µm was estimated (Bastacky et al 1995). The primary regulators of the
alveolar liquid layer are ATII cells. For many years, ATI cells have been believed to
represent only a physical barrier, but recently studies showed that ATI cells express
molecules crucial for fluid reabsorption and that they can as well actively contribute to
fluid clearance (Johnson et al., 2002).
The regulation of alveolar surface liquid volume is controlled by an orchestrated active
absorption and secretion of Na+- and Cl-- ions, which create an osmotic driving force for
water to follow. Key elements are epithelial Na+- channels (ENaCs) and Cl- - channels
for example cystic fibrosis transmembrane conductance regulator (CFTR), located in
the apical surface of alveolar epithelial cells and the Na+,K+-ATPase, a cation
transporter located in the basolateral membrane.
Especially in the fetal lung Cl--transport is extremely important, since it drives
distention of the lung, enabling growth and development (Olver and Strang, 1974). The
mechanism is independent of pulmonary vascular filtration (Carlton et al., 1992a)
indicating that it is taking place at the alveolar epithelium. This process is described to
be secondary active, with Cl- being cotransported into the cell by the Na+,K+,2Clcotransporter (NKCC) located in the basolateral membrane and induced by the
electrochemical Na+-gradient created by the Na+,K+-ATPase (Carlton et al., 1992b;
Cassin et al., 1986). This results in an excess of Cl- in the cell that is then secreted
apically into the airspace. Around the time of birth, the predominance of Cl--secretion
fades and Na+-absorption takes over to clear the lung from excess fluid.
The alveolar Na+-transport is mediated by ENaCs and the Na+,K+-ATPase. As
mentioned above, the Na+,K+-ATPase pumps Na+-ions out of the cell in exchange for
6
Introduction
7
K+, more precisely three Na+-ions and two K+-ions. The result is a Na+-gradient
between the airspace and the interstitium. Early studies in the rabbit urinary bladder,
another tight epithelium exhibiting Na+-transport similar to the lung, investigating the
properties of the apical and the basolateral membrane conclude that the Na+conductivity of the apical membrane is limiting the transcellular Na+-flux (Lewis et al.,
1977). This also applies to the lung (Canessa et al., 1994).
Figure 1 Scheme of alveolar Na+-transport
The Na+,K+-ATPase removes Na+ from the cytosol, creating a gradient for Na+ to enter the apical side of the
alveolar cells. This is happening both in ATI and ATII cells. Water is following the osmotic gradient that is
created. The result is a reduction of alveolar surface liquid.
Introduction
1.2. Deg/ENaC superfamily
ENaCs have first been reported from studies using frog skin and toad urinary bladder
that were mounted in an Ussing chamber to measure transepithelial ion transport. ENaC
or ENaC-like Na+-channels are widely distributed among different animal species.
Molecules belonging to the Degenerin/ENaC-superfamily have been identified from
different epithelia in nematodes (Lai et al., 1996), flies, several species of rodents
(Canessa et al., 1994; Letz et al., 1995), frog, chicken (Goldstein et al., 1997), cow
(Fuller et al., 1995), lungfish (Li et al., 1995) and man (Fronius et al., 2010) underlining
the evolutionary importance of this protein family.
1.2.1. Common structure
All members of the Deg/ENaC superfamily share invariable topology: Two
transmembrane domains are connected with a large extracellular loop, that contains
cysteine-rich domains (Renard et al., 1994). The N- as well as the C-termini are located
in the cytoplasm of the cells and represent only a small fraction of the full-length
protein (Mano and Driscoll, 1999). Many proteins including ENaCs assemble in homoor hetero-multimeric complexes with varying composition (Firsov et al., 1998; Kosari et
al., 1998; Snyder et al., 1998).
1.2.2. Various functions
The functions mediated by members of the Deg/ENaC superfamily are as diverse as
their regulation. Touch sensation in C. elegans has been shown to be dependent on the
mechanosensory abnormality proteins (MEC-4 and MEC-10) that assemble in a
heteromeric complex containing at least two copies of each subunit (Hong and Driscoll,
1994; Huang and Chalfie, 1994). Proprioception is another sense that involves members
of that family (uncoordinated (UNC-8) (Tavernarakis et al., 1997) and UNC-105
(Garcia-Anoveros Garcia Liu)).
Other functions of the Deg/ENaC superfamily involve neurodegeneration (Chalfie and
Wolinsky, 1990) and sensing of protons (Chen et al., 1998). pH-dependency of ASIC
channels in vitro required very low pH and the result was a rapidly inactivating cation
current (Lingueglia et al., 1997; Waldmann et al., 1997).
Most important for the elaborated study is the regulation of salt absorption controlled by
ENaCs located in epithelial cells of the kidney, colon and lung. As published by Bentley
in 1968 amiloride is a very potent inhibitor of ENaC (Bentley, 1968). Several types of
8
Introduction
channels have been described, all of which are sensitive to amiloride namely highly
selective channels (HSC) for Na+ and Li+ with a low conductance of 5 pS, and low
selective channels (LSC) with a conductance of 9 pS and 28 pS (Palmer, 1992).
1.2.3. Epithelial Na+-channels
Four subunits have been identified in mammals, αβγδ-ENaC. The first subunit that was
identified in the rat colon was α-ENaC (Canessa et al., 1993). When heterologously
expressed in Xenopus oocytes, it showed characteristics that were found previously of
channels expressed in native epithelia, namely high amiloride-sensitivity, high
permeability for Li+ and no K+-conductivity. The only difference was that the currents
were much smaller, compared to injection of whole colon RNA. Subsequently the
authors searched for further subunits and found two more subunits, β- and γ-ENaC
which, when expressed individually, did not form Na+-conducting, amiloride-inhibitable
channels (Canessa et al., 1994). When β- and γ-ENaC were coexpressed together with
α-ENaC, the current was more than 100 fold higher, compared to α-ENaC alone
(Canessa et al., 1994), suggesting that α-ENaC is the pore-forming subunit whose
properties are modulated by β- and γ-ENaC.
δ-ENaC has been identified in 1995. This subunit shares some features with, but also
exhibits some important differences to α-ENaC: Similar to α-ENaC, δ-ENaC expressed
individually forms amiloride-sensitive channels and the current is increased by two
orders of magnitude, when β- and γ-ENaC are coexpressed. Different to α-ENaC it is
more permeable for Na+, than for Li+ and the sensitivity for amiloride was
approximately 30 times higher (Waldmann et al., 1995). Closely related to δ-ENaC,
functionally as well as structurally, is ε-ENaC a subunit that is exclusively expressed in
the clawed frog X. laevis (Babini et al., 2003).
Although the structure of all ENaC-subunits is quite similar, the tissue expression
pattern differs markedly. α- and β-ENaC are predominantly expressed in lung and
kidney, whereas γ- and δ-ENaC are found in a variety of epithelial and non-epithelial
tissues of different organs (Ji et al., 2012). δ-ENaC is expressed in heart, liver, brain and
lung, but also found in pancreas, skeletal muscle and blood leukocytes (Su et al., 2004;
Waldmann et al., 1995).
The functions of β- and γ-ENaC are not as clear as for the other subunits. β-ENaC has
been postulated to regulate trafficking of ENaC complexes to the plasma membrane. Its
Clara cell specific overexpression in the mouse resulted in increased Na+-transport of
9
Introduction
tracheal tissue that caused a cystic-fibrosis-like phenotype with mucus accumulation
and postobstructive enlargement of distal airspaces, while overexpression of α- and γENaC did not affect Na+-transport (Mall et al., 2004). An explanation for this
phenomenon might be that β-ENaC is normally the least abundant and thus limiting
subunit in the lung (Farman et al., 1997; Talbot et al., 1999; Yue et al., 1995). Genetic
knockdown studies on the other hand showed, that the fluid clearance in the lung is
impaired, when β-ENaC expression is reduced (Randrianarison et al., 2008).
Stochiometry of ENaC is still under debate. Generally it is believed that ENaC is a
heterotetrameric complex composed of two α-, one β- and one γ-subunit (Firsov et al.,
1998). Using the very sophisticated approach of atomic force microscopy a trimer
composed of one copy each of α-, β- and γ-ENaC or even a trimer-of-trimers was
described (Stewart et al., 2011). Considering the discovery of δ- and ε-ENaC which
might replace α-ENaC or could even be added to the conventional heteromeric complex,
various subunit assemblies in native tissues and different in vitro models might explain
the great variations that can be observed studying ENaC function.
1.2.4. Regulation
Since the control of Na+-transport is crucial to maintain proper water and salt
homeostasis, ENaC function is tightly regulated on different levels by a variety of
factors.
Regulation of gene expression is limited to long term regulation and less important than
acute effects. Significant changes of the single channel conductance due to regulatory or
genetic changes could not be demonstrated. The predominant elements of acute ENaC
regulation are the number of channels in the membrane (N) and their open probability
(P0) which indicates the ratio of time the channel is in the “open” as opposed to the
“closed” conformation (Butterworth, 2010).
Only a very small portion of all ENaC proteins is located in the plasma membrane. The
vast majority is located in trafficking vesicle pools (Hanwell et al., 2002). When
introduced into the plasma-membrane ENaCs form near-silent channels, which require
activation by proteolytic processing of the α- and γ-subunit to be fully functional
(Carattino et al., 2008; Sheng et al., 2006).
Stimuli involved in ENaC regulation include hormones, especially aldosterone (Lee et
al., 2008; Masilamani et al., 1999) and insulin (Blazer-Yost et al., 2003; Deng et al.,
2012) as well as estrogen and progesterone (Gambling et al., 2004).
10
Introduction
Also, anorganic chemicals regulate ENaC activity. Na+ itself, for example, has a dual
role on ENaC function. Intracellular Na+ has been shown to reduce proteolytic
activation of the channel by rendering its cleavage sites inaccessible to proteases by
changing the conformation of the complex. This process happens relatively slow (within
minutes) and is called “feedback inhibition”. The physiologic function is to prevent the
cells from Na+ overload and thus cell swelling (Knight et al., 2008; Komwatana et al.,
1996; Uchida and Clerici, 2002).
Extracellular Na+ on the other hand triggers “Na+ self-inhibition”. When the
extracellular Na+-concentration is increased rapidly, ENaC activity is impaired. In
contrast to the feedback inhibition this response is happening within seconds, induced
by a decrease of the open probability P0. This process can not be explained by saturation
of Na+-transport, because increasing the Na+-concentration stepwise does not result in a
comparable inhibition (Fuchs et al., 1977). The molecular principle of “Na+ selfinhibition” has been shown to involve a conformational change in the proximal part of
the extracellular loop of α- and ε-ENaC (Babini et al., 2003). Also other stimuli
independent of Na+, like acidity, seem to act on ENaC via the “Na+ self-inhibition” as
has been described for human ENaC (Collier and Snyder, 2009).
Oxygen tension is an important factor in the conversion of non-seletive to high-selective
channels. Culture of ATII cells with only 5 % O2 resulted in predominantly nonselective channels. However after 2 h with 95 % O2 the majority of channels was highly
selective (Jain et al., 2008).
Chlorine has also been reported to reduce ENaC expression in the membrane and its
activity, in the mouse as well as in isolated ATII cells (Lazrak et al., 2012).
Further stimuli modulating ENaC activity are redox state (Downs et al., 2013; Helms et
al., 2008) and physical factors such as shear stress (Fronius et al., 2010).
1.2.5. Signaling pathways
There are multiple signaling pathways that are known to regulate ENaC. Several
steroids have been implicated in the translational regulation of ENaC, mediated
probably via interaction with the glucocorticoid responsive element (GRE) located
upstream of the promotor of α-ENaC (Dagenais et al., 2008). Recent studies show that
translation of ENaC is modulated also by miRNA (Tamarapu Parthasarathy et al.,
2012).
11
Introduction
Interleukin-1β (IL-1β), an important cytokine in ARDS, was shown to reduce ENaC
mRNA levels via the P38 mitogen-activated protein kinase (MAPK) (Roux et al.,
2005). Another member of the MAP-kinase family, the extracellular signal-regulated
kinase (ERK), is also a regulator of ENaC and has been associated with phosphorylation
of β- and γ- ENaC. This phosphorylation is likely to enhance interaction with the E3ubiquitin-ligase neural precursor cell expressed developmentally down-regulated
protein 4-2 (Nedd4-2) (Yang et al., 2006), a protein that directly attaches the small
molecule ubiquitin to specific target proteins, leading to endocytosis and subsequent
degradation (Kabra et al., 2008). Ubiquitination of ENaC is a highly complex
mechanism to differentially regulate protein stability in the cytoplasm and in the plasma
membrane. Nedd4-2 is the most prominent E3-ligase known to regulate ENaC, but there
is evidence for other E3-ligases that interact with ENaC (Downs et al., 2013). Upon
activation Nedd4-2, preferably its domains WW2 and/or WW3 (Itani et al., 2009), binds
to the highly conserved PY-motifs, located at the C–terminus of all ENaC subunits,
leading to ubiquitination of lysines located at the N-terminus of ENaC subunits,
rendering proteins located in the cytosol susceptible for proteasomal degradation,
whereas complexes containing αβγ-ENaC located in the plasma membrane appear to be
targeted for lysosomal degradation (Staub et al., 1997).
Another way of Nedd4-2 mediated ENaC regulation is a conformational change of the
extracellular domain upon ubiquitination that impairs proteolytical processing, thus
preventing activation of immature channels (Ruffieux-Daidie and Staub, 2010).
Several pathways seem to converge in ubiquitination. The important effector of
aldosterone the serum- and glucocorticoid regulated kinase (SGK1) (Debonneville et
al., 2001; Lee et al., 2008) but also inhibitor of nuclear factor kappa-B kinase subunit β
(IKKβ) (Edinger et al., 2009) and the metabolic sensor adenosine-monophosphateactivated kinase (AMPK) (Almaça et al., 2009) phosphorylate Nedd4-2, disrupting its
interaction with ENaC.
12
Introduction
1.3. State of the art
Elevated CO2 levels have been shown to reduce alveolar epithelial function
independently of pH by downregulation of the Na+,K+-ATPase (Briva et al., 2007).
Further studies elucidating the underlying pathway identified AMPK to be activated
during hypercapnia and to be involved in endocytosis of the Na+,K+-ATPase (Vadász et
al., 2008). The exact mechanism upstream of AMPK is incompletely understood,
especially the sensor for CO2 is still unknown.
AMPK phosphorylates Nedd4-2 thus promoting its translocation to the membrane
where it ubiquitinates individual or all ENaC subunits (Almaça et al., 2009; Bhalla et
al., 2006; Myerburg et al., 2010). Ubiquitinated channels are then retrieved from the
membrane and degraded by either the lysosome or the proteasome (Lazrak et al., 2012;
Malik et al., 2006).
A pharmacological study elucidating the role of several AMPK-activators on alveolar
epithelial Na+-transport provides evidence for a differential effect of AMPK on ENaC
and the Na+,K+-ATPase (Woollhead et al., 2007). Specific modulation of ENaC and the
Na+,K+-ATPase by AMPK could be relevant in the resolution of pulmonary edema.
Detection of ENaC proteins in the lung is difficult. Antibodies against endogenous
proteins are poor and often recognize either the full length form or the cleaved form of
α- and γ-ENaC (Boncoeur et al., 2009; Downs et al., 2013; Jain et al., 1999). Even if
both forms are recognized, antibodies against each cleaved fragment produce
inconclusive protein sizes (Albert et al., 2008). Many investigators, especially in the
field of nephrology are working with overexpression of modified ENaC-subunits that
can be recognized better than the endogenous proteins, but reliable systems for
detection of ENaC protein in lung cells are not well established.
13
Introduction
14
The aim of the present study was initially to decipher the effect of CO2 on ENaC
function according to the working hypothesis illustrated in Figure 2. Due to
insufficiencies of the available systems a second aim was added later:
Establishment of a system to detect and immunoprecipitate immature and mature ENaC
proteins in lung epithelial cells.
Figure 2 Proposed mechanism of CO2 regulated endocytosis of ENaC
CO2 is indirectly activating AMPKα1 which is phosphorylating Nedd4-2. Nedd4-2 attaches ubiquitin (Ub) to membrane
bound ENaC to target it for endocytosis and subsequent degradation or trafficking back to the membrane.
Materials and Methods
15
2. Methods
2.1. Cell culture
2.1.1. Culture of A549 cells
A549 cells show characteristics of alveolar epithelial cells and can be transfected easily.
Cells were grown in 100 mm cell culture dishes. The full medium contained DMEM (4.5
g/l glucose incl. stable L-glutamine) and 10 % [v/v] fetal bovine serum (FBS). No
antibiotics were used and the cells were maintained in a humidified incubator at 37 °C, 5 %
CO2, 100 % relative humidity.
For treatment with high CO2-levels the medium had to be modified to compensate for the
influence of CO2 on the pH. For that 3 ml of basal DMEM (4.5 g/l glucose incl. stable Lglutamine), 1 ml F12-supplement and 0.5 ml of Tris-solution (0.5 M, pH 7.3 or pH 10 for
normocapnia and hypercapnia respectively) and the solutions incubated in 5 % respectively
18 % CO2 cell culture incubators to achieve an identical pH of 7.4 and PO2 but a PCO2 in the
medium of either 40 or 110 mmHg, as published previously (Briva et al., 2007; Vadász et
al., 2008).
2.1.2. Transfection of A549 cells
For transfection of plasmid DNA 6 x 105 A549 cells were plated on 60 mm cell culture
plates in 3 ml of full medium. The following day 12 µl Lipofectamine 2000 were diluted in
500 µl DMEM, incubated for 5 min, then 4 µg plasmid-DNA was added and the mixture
incubated for 30 min. The transfection mixture was then directly added to the cells without
aspiration of the medium. After transfection no media change was performed, since
cytotoxicity was minimal and higher expression rates were obtained. Experiments were
conducted 18-24 h after transfection.
Transfection with siRNA was performed using Lipofectamine RNAiMAX. 6 x 105 A549cells were plated on a 60 mm cell culture dish in 2 ml DMEM. The next day cells were
starved with preequilibrated DMEM (1.5 g/l glucose) without serum for 30 min. Per
reaction, 17 µl siRNA and 17 µl Lipofectamine RNAiMAX were diluted in 250 µl
OptiMEM medium each, mixed by pipetting and combined. After a 30 min incubation
time, medium was aspirated from the cells and the 500 µl transfection solution applied to
the cells. During a period of 4 h the cells were agitated every 15 minutes, before 1 ml of
16
OptiMEM was added. Cells were replated and transfected the next day with ENaCplasmids as stated above in this paragraph.
2.1.3. Culture of H441 cells
To elucidate the effect of hypercapnia on epithelial Na+-transport Ussing chamber
experiments were conducted on H441 cells. H441 cells, in contrast to A549 cells, form
polarized monolayers and are established as a model for investigation of Na+-transport in
alveolar epithelial cells (Albert et al., 2008; Brown et al., 2008; Ramminger et al., 2004).
The basal medium for subculturing H441 cells was RPMI 1640 incl. L-glutamine, 10 %
[v/v] FBS, 100 IU/ml Penicillin, 100 µg/ml Streptomycin, 1 mM Na+-Pyruvat, 5 µg/ml
Insulin, 5 µg/ml Transferrin, 5 µg/ml Na+-Selenit.
For functional measurements cells were seeded on snapwell-inserts with a growth area of 1
cm2. The following day the medium on the apical side was aspirated so that the apical
compartment was exposed to air. Furthermore the medium on the basolateral side was
supplemented with 200 nM dexamethasone. Experiments were performed 5-7 days later
with the medium replaced every two days.
2.1.4. Optimization of H441 cell transfection
Transfection of H441 cells by Lipofectamine 2000 is not efficient, so another system had
to be used. A new method designed for primary cells and hard-to-transfect cell lines is
Nucleofection, a modified electroporation technique. Basic principle is the formation of
pores in the plasma membrane as well as in the nuclear envelope by specifically combining
solutions with different ionic compositions with electrical impulses of different
frequencies, durations and intensities. The best combination has to be established
empirically for every cell-type.
Nucleofection was performed using a Lonza 4D-Nucleofector and preselected cell typespecific solutions. For optimizing Nucleofection of cells in suspension H441 cells were
cultured for 24 h. Cells were then trypsinized, counted and the appropriate amount of cells
(1.5 x 105 cells per reaction) centrifuged at 90 g for 10 min. The supernatant was discarded,
the cells resuspended in Nucleofection mixture (20 µl reaction: 16.4 µl Solution SF, 3.6 µl
of supplement + 0.4 µg pMaxGFP Vector) and transferred to the Nucleofection cuvette.
Nucleofection was conducted in the X-Unit of the device. After Nucleofection 80 µl of
prewarmed medium was added into the cuvette to resuspend the cells and 50, 25 and 12.5
µl of cell-suspension were plated in 150, 175 and 187.5 µl respectively of prewarmed
17
medium in a 96-well plate. 24 h after Nucleofection efficiency and viability were estimated
and the best program selected.
2.1.5. Transfection of H441 cells
For transfection of H441 cells in suspension the 4D-Nucleofector System and the SF Cell
Line 4D-Nucleofector X Kit was used.
Cells were plated such, that they reached not more than 50 % confluence 24 h later. The
next day cells were washed with PBS, detached from the culture plate with trypsin,
counted and the desired amount of cells (1 x 106 cells per reaction) was centrifuged at 90 g
for 10 min. The supernatant was discarded and the cells were resuspended in nucleofection
solution (82 µl solution SF, 18 µl supplement), combined with the desired amount of
nucleic acid, e.g. 2 - 4 µg pMaxGFP Vector and transferred to the Nucleofection cuvette.
The program used for Nucleofection was CM 138. Cells were then incubated at room
temperature for 10 min, diluted in app. 300 µl of prewarmed medium and plated. For
transwell supports and 60 mm cell culture dishes 1 x 106 cells were plated, 0.5 x 106 cells
for 35 mm dishes. Analysis was conducted 24 - 48 h later.
2.1.6. Isolation of primary rat alveolar epithelial cells
Primary rat alveolar epithelial type II cells (ATII) were isolated as described previously
(Buchäckert et al., 2012; Dobbs, 1990) and cultured in DMEM with 4.5 g/l glucose, 10 %
[v/v] FBS and 100 IU/ml Penicillin, 100 µg/ml Streptomycin.
2.1.7. Establishment of ATII cell transfection
Transfection of ATII cells was performed by Nucleofection using a Lonza 4DNucleofector and preselected cell type-specific solutions as recently published (Grzesik et
al., 2013). For optimizing Nucleofection of primary ATII cells in suspension, cells were
cultured for 24 h. Cells were then trypsinized, counted and the appropriate amount of cells
(1.5 x 105 cells per reaction) centrifuged at 90 g for 10 min. The supernatant was discarded,
the cells resuspended in Nucleofection mixture (20 µl reaction: 16.4 µl Solution P1 or P3,
3.6 µl of supplement + 0.4 µg pMaxGFP Vector) and transferred to the Nucleofection
cuvette. Nucleofection was conducted in the X-Unit of the device. After Nucleofection 80
µl of prewarmed medium was added into the cuvette to resuspend the cells and 50, 25 and
12.5 µl of cell-suspension were plated in 150, 175 and 187.5 µl respectively of prewarmed
18
medium in a 96-well plate. 24 h after Nucleofection efficiency and viability were estimated
and the best program selected.
Further optimization revealed no loss of transfection efficiency, when using up to 3.5 x 106
cells per 100 µl reaction. Attempts to transfect freshly isolated cells resulted in significant
cell death.
Some cell types that are cultured in high-calcium-medium (e.g. DMEM) require a recovery
step after transfection to prevent an influx of calcium through the not yet closed pores in
the plasma-membrane. For that step low-calcium-medium (e.g. RPMI 1640) is added
directly to the Nucleofection cuvette and the cells are incubated at 37 °C for 10 minutes.
This step proved not to be necessary for ATII cells. Also, removal of Nucleofection
solution after transfection by diluting the cells in preequilibrated medium and subsequent
centrifugation did not result in higher viability, nor transfection efficiency.
2.1.8. Final protocol for the Nucleofection of ATII cells
For the optimized protocol freshly isolated rat AT II cells were plated in 60 mm cell
culture dishes to recover from the isolation. The next day cells were washed with PBS,
detached from the culture plate with trypsin, counted and the desired amount of cells (3.5 x
106 cells per reaction) was centrifuged at 90 g for 10 min. The supernatant was discarded
and the cells were resuspended in nucleofection solution (82 µl solution P3, 18 µl
supplement), combined with the desired amount of nucleic acid, e.g. 3-5 µg pMaxGFP
Vector and transferred to the Nucleofection cuvette. The program used for Nucleofection
was EA-104. Cells were then incubated at room temperature for 10 min, diluted in app.
300 µl of prewarmed medium and plated. For transwell supports and 60 mm cell culture
dishes 3.5 x 106 cells were plated, 2 x 106 cells for 35 mm dishes. Analysis was conducted
48 h later (day 3 after isolation).
2.1.9. Flow cytometry
For determination of transfection efficiency, cells were washed with PBS, trypsinized,
centrifuged at 90 g and resuspended in FACS buffer (PBS without Ca2+ and Mg2+, 7.4 %
EDTA, 0.5 % FBS, pH 7.2). To evaluate viability, cells were stained with 7-AAD (1:10)
and incubated for 5 min prior to FACS analysis which was conducted with a LSR Fortessa
and analyzed by FACS Diva Software.
19
2.1.10. Fluorescence microscopy
GFP expression of transfected cells was visualized by fluorescence microscopy. Living
cells were imaged in medium using a fluorescence microscope (Leica DMIL), a camera
(Leica DFC 420C) and the software Leica application suite 330.
2.1.11. Cytotoxicity assay
Early cell death was evaluated by release of lactate-dehydrogenase (LDH) into the
medium. Cells were transfected and plated on permeable supports as described above. The
medium was collected at 4 h, replaced by fresh medium and collected again 24 h after
transfection. The assay was performed as instructed by the manufacturer. Minimal cell
death was measured in non treated cells, maximal cell death was determined by lysing cells
with 1 % Triton X 100.
2.2. Ussing chamber measurements
Ussing chamber experiments were conducted on H441 cells. For those experiments cells
were plated confluent on snapwell permeable supports. One day after plating, medium was
removed from the apical side to culture the cells at liquid-air-interface and dexamethasone
200 nM was added to the medium. Measurements were conducted on day 7 - 9 after
plating.
Experiments were performed using a custom built Ussing chamber setup. The electrical
signal was recorded and amplified by a voltage-clamp-amplifier (custom built; Institut for
animal physiology Gießen), converted from analog to digital by an AD/DA-converter
(MacLab Interfaces) and recorded by a two-channel chart-strip recorder (Kipp & Zonen,
Netherlands) plus recorded digitally using an Apple-PC, (Macintosh, Apple) and the
software Chart (Version 3.6.3, MacLab). Electrical interference was neutralized with a 50
Hz filter that was included in the software. The relevant data where also noted manually.
The regular perfusion solution contained in mM: NaCl 130, KCl 2.7 KH2PO4 1.5, MgCl2 :
0.5, CaCl2, D-glucose 10. Unless otherwise stated, 55 mM Tris base was added. For
normocapnia (pCO2 ~ 40 mm Hg) and hypercapnia (pCO2 > 100 mm Hg) the solutions
were continuously bubbled with 5 % CO2, 21 % O2, 74 % N2 and 20 % CO2, 21 % O2, 59
% N2 respectively and the pH adjusted accordingly to obtain a final pH of about 7.4.
20
AD converter + computer
electrodes
perfusion
cells
apical
basolateral
Figure 3 Scheme of the custom build Ussing chamber.
Both chambers of the setting were perfused seperately so it was possible to specifically target the apical or
the basolateral compartment.
21
2.3. Real-time rtPCR
To study the expression of endogenous ENaC subunits on the mRNA level, real-time
rtPCR experiments were performed. After incubating the cells in normo- versus
hypercapnic conditions for 6 resp. 24 h, cells were lysed and mRNA isolated using the
RNeasy-Kit according to the instructions supplied by the manufacturer. The mRNA was
reverse transcribed (iScript cDNA Synthesis Kit (Table 2) and PCR performed with
iTaqTM Sybr Green Supermix with ROX. The reaction composition is depicted in Table
3. The expression of the housekeeeping gene hypoxanthin-phosphoribosyl-transferase
(HPRT) was used to normalize the data.
Calculation of data:
ΔCt = Ct housekeeping gene - Ct target gene
ΔΔCt = Ct CO2 – Ct control
Table 2 Composition cDNA synthesis (20 µl reaction)
component
volume
Reaction buffer (5x)
4 µl
reverse transcriptase
1 µl
RNA (1µg)
x µl
H2O
to 20 µl
Table 3 Composition real-time PCR (25 µl reaction)
component
volume
iTaq Sybr Green Supermix with ROX
12.5 µl
cDNA (1:5 diluted)
2.0 µl
Primer forward
0.5 µl
Primer reverse
0.5 µl
H2O
9.5 µl
Table 4 Real time PCR conditions
initial denaturation
95°C
2:00 min
denaturation
95°C
1:15
sec
primer annealing + elongation
55°C
0:30
sec
4°C
-
cool down
35 cycles
22
Following primers were used for real-time PCR:
Table 5 Real-time PCR Primer
gene
primer
primer-sequence
reference-sequence
α ENaC
hscnn1a_F
5`-acttcagctaccccgtcagc-3´
NM_001038
hscnn1a_R
5`-gagcgtctgctctgtgatgc-3´
hscnn1b_F
5`-gcaccgtgaatggttctgag-3´
hscnn1b_R
5`-cggatcatgtggtcttggaa-3´
hscnn1d_F
5`-cagcatccgagaggacgag-3´
hscnn1d_R
5`-aggagcaggtctccaccatc-3´
hscnn1g_F
5`-gctgcctactcgctccagat-3´
hscnn1g_R
5`-ttcctggacaaaggctcgat-3´
HPRT_human_F
5`-cctggcgtcgtgattagtga-3´
HPRT_human_R
5`-atggcctcccatctccttc-3´
β ENaC
δ ENaC
γ ENaC
HPRT
NM_000336.2
NM_001130413.3
NM_001039
NM_000194.2
2.4. Generation of genetically modified ENaC-constructs
The expression levels of ENaC-subunits are generally low. Furthermore the majority of
ENaC proteins is located in submembranous vesicles and in the endoplasmatic reticulum,
making the detection of functional ENaC proteins in the plasmamembrane even more
challenging (Hanwell et al., 2002). To enhance the expression α, β and γ-ENaC were
cloned into expression vectors. The generated constructs were genetically modified:
Epitope-tags were added to increase the recognition for commercially available antibodies.
eYFP-α-ENaC-Flag, β-ENaC-V5, and Myc-γ-ENaC-HA were generated (see Table 6). βENaC was the first vector generated and cloned from the human cellline A549 (see 2.1.1).
Later, α- und γ-ENaC were cloned from previously generated plasmids (pTNT-Oocyte
expression vector; (Fronius et al., 2010)) tagged with the respective epitope-tags and
ligated into expression vectors.
23
Table 6 Cloning primers and genetic modifications
protein
primer
primer sequence
destination-
tag
vector
α ENaC
α ENaC
SCNN1A_for2
5`-catggaggggaacaagct-3‘
SCNN1A_rev2
5´-ccttggtgtgagaaacctctcc -3`
SCNN1A_for3
5`-gaattcaatggaggggaacaagctggagg-3‘
SCNN1A_rev
5´-ggatcccttgtcatcgtcatccttgtaatcggg`
pE-eYFP
eYFP
Flag
1
cccccccagaggac-3`
β ENaC
SCNN1Bfor
5`-ctcggatccacatgcacgtgaagaagtacct-3`
pcDNA3.1V5/
V5
Hyg
γ ENaC
1
SCNN1Brev
5`-gcactcgaggatggcatcaccctcactgt-3`
SCNN1G_forHA
5`-aggcccgaattcatggcacccggagagaagat-3`
SCNN1G_revHA
5`-gtagccggtaccgagctcatccagcatctggg-3`
SCNN1G_formyc
5`-gagatcggatccaatggcacccggagagaagat-3`
SCNN1G_revmyc
5`-ccccccctcgagttaagcgtaatctggaacat-3`
Highlighted is the sequence for the FLAG-epitope-tag that was added by PCR
pCMV-HA-C
HA
pCMV-tag 3
Myc
24
2.4.1. Subunit-specific cloning procedure
2.4.1.1. β-ENaC
For the cloning of b-ENaC, mRNA was isolated from A549 cells and reverse transcribed
into cDNA. Amplification of the coding sequence with the appropriate restriction sites
attached was performed with Pfu-Ultra DNA polymerase. The sequence of the primers
used is provided in Table 6. The composition of the PCR-mix is listed in Table 7, the
cycler-conditions are given in
Table 8.
Table 7 PCR reaction β-ENaC (25 µl)
component
volume
H2O
to 25.0 µl
buffer
2.5 µl
dNTP
0.4 µl
primer forward
0.5 µl
primer reverse
0.5 µl
cDNA
1.0 µl
Pfu DNA polymerase
1.0 µl
Table 8 PCR conditions for cloning of β-ENaC
97°C
10:00 min
denaturation
95°C
1:30 min
primer annealing
65°C
0:30 min
elongation
72°C
4:00 min
final elongation
72°C
10:00 min
cool down
4°C
35 cycles
-
The PCR-product was analyzed in a 1 % agarose-TAE-buffer gel and the fragment size
compared to the expected size of the amplicon.
The PCR product was purified using the Wizard® SV Gel and PCR Clean-Up System. The
product and the vector pcDNA3.1 V5/Hyg were then digested with XhoI und BamHI at
25
37°C over night (Table 9) and again purified with the PCR Clean-up System. Complete
digestion was verified by electrophoretical determination of the fragment size.
Table 9 Restriction digestion for b-ENaC
component
volume
H2O + DNA
40.0 µl
enzymebuffer B 10x
5.4 µl
BSA acetylated
5.4 µl
XhoI
1.4 µl
BamHI
1.4 µl
The digested vector and a two-fold excess of insert were ligated over night at 4°C with T4
ligase. As a negativ control the ligation was performed without insert.
Table 10 Ligation reaction
component
volume
H2O
to 20 µl
buffer 10x
2 µl
vector
100 ng
insert
140 ng
T4 ligase (3 U/µl)
1 µl
2.4.1.2. α-ENaC
Since the α-subunit of ENaC undergoes proteolytical processing during maturation, the
two major fragments were modified to contain individual epitope tags. The primers needed
for the modification were relatively long with a brief overlap at the coding region of the
protein, resulting in special conditions for the PCR. Increased difficulties arose from the
sequence of the human α-ENaC being rich in the nucleotides guanine and cytosine,
aggrevating the cloning process.
Therefore the plasmid was cloned in two steps. First the coding region plus some overhang
was amplified from a preexisting plasmid (pTNT-Oocyte expression vector (Fronius et al.,
2010)) with the newly distributed Q5 polymerase which has a higher fidelity and acuracy
compared to the Pfu-polymerase used to clone the β-subunit. The primers were designed to
only amplify the coding region and some overhang. The PCR product of this reaction was
26
then used as a template for the next reaction, that was performed with primers that included
restriction sites and the tag to reduce non-specific annealing. The destination vector
pEYFP-C1 contained the epitope-tag eYFP at the N-terminus. To add another tag at the Cterminus of the fusion protein the sequence for the FLAG-tag (5´-gat tac aag gat gac gat
gac aag-3`) was incorporated in the reverse-primer between the coding sequence and the
stop codon (see Table 6 highlighted).
Table 11 PCR reaction for α-ENaC (25 µl)
component
volume
H2O
15.75 µl
buffer
5.00 µl
dNTP
0.50 µl
primer forward
1.25 µl
primer reverse
1.25 µl
cDNA
1.00 µl
Q5 DNA polymerase
0.25 µl
Table 12 First PCR condition for cloning of α-ENaC
95°C
0:30 min
denaturation
95°C
0:10 min
primer annealing
65°C
0:30 min
elongation
72°C
2:10 min
cool down
4°C
35 cycles
-
Table 13 Second PCR condition for cloning of α-ENaC
95°C
0:30 min
denaturation
95°C
0:10 min
primer annealing + elongation
72°C
2:10 min
cool down
4°C
35 cycles
-
The PCR product and the vector were purified and digested with fast-digest restriction
enzymes at 37°C for 15 minutes (see Table 14) and purified again for ligation.
27
Table 14 Restriction digestion α-ENaC (40 µl volume)
component
volume
H2O + DNA
32 µl
buffer FDgreen 10x
4 µl
EcoRI
2 µl
BamHI
2 µl
For the ligation the insert and vector were mixed at a molar ratio of 3:1 and ligated with the
T4 ligase kit. As a negative control ligations without vector were performed.
Table 15 Ligation for α-ENaC T4 ligase kit (20 µl)
component
volume
H2O
to 20 µl
buffer 10x
2 µl
vector
100 ng
insert
140 ng
T4 ligase (3 U/µl)
1 µl
2.4.1.3. γ-ENaC
Cloning of the double-tagged γ-ENaC was also performed in two steps: The coding
sequence was first amplified, ligated in the vector pCMV-HA-C, containing the c-terminal
HA-tag and transformed into bacteria (see 2.4.2). Successful transformation was controlled
by colony-PCR with the primer-pair SCNN1G_for/revmyc. This step provided two
benefits: Since one primer was designed to align to a region on the first destination-vector
the stringency for the screening was increased. Additionally, the sequence of the plasmid
containing the HA-tag was included in the PCR-fragment, so that the PCR product could
not only be visualized electrophoretically, but also purified and ligated directly into the
second vector pCMV-tag 3 that added a N-terminal Myc-tag. Both PCR-reactions were
performed with the conditions given in Table 11 and Table 13. Restriction digestion was
performed according to Table 14, ligation as described in Table 15.
28
2.4.1.4. Site directed mutagenesis α-ENaC
Two single nucleotide polymorphisms (SNP) were included in the original α-ENaC
plasmid that caused a change in the aminoacid sequence (A334T and T663A) compared to
the reference sequence NM_001038. Although they were reported earlier (rs11542844 and
rs2228576), site directed mutagenesis was performed to eliminate possible effects on the
biology of the protein.
Table 16 Primers for site directed mutagenesis
primer
primer sequence
A2365G_for
5`-gtccctgatgctgcgcgcagagcagaatgacttc-3`
A2365G_rev
5`-gaagtcattctgctctgcgcgcagcatcagggac-3`
G3352A_for
5`-ggggccagttcctccacctgtcctctggg-3`
G3352A_rev
5-cccagaggacaggtggaggaactggcccc-3`
The mutagenesis was performed with the QuikChange Site-Directed Mutagenesis Kit. The
reaction conditions are described in Table 17 and Table 18.
Table 17 QuikChange site directed mutagenesis reaction
component
volume
H2O
to 25.0 µl
buffer
2.5 µl
dNTP
0.5 µl
primer forward
62.5 ng
primer reverse
62.5 ng
Pfu Turbo DNA polymerase (2.5 U/µl)
1.0 µl
template ( ~ 20 µg plasmid)
1.0 µl
Table 18 QuikChange site directed mutagenesis thermal profile
95°C
0:30 min
denaturation
95°C
0:30 min
primer annealing
55°C
1:00 min
elongation
68°C
7:00 min
cool down
4°C
-
16 cycles
29
The parental, non modified vector was digested with 1 µl DpnI at 37°C for 1 h and the
reaction with now only the modified plasmids directly transformed into XL1-Blue
supercompetent cells, supplied with the QuikChange Kit. For that the cells were thawed on
ice and a 25 µl aliquot transferred to a prechilled 14 ml polypropylene tube. One µl of the
digested DNA was added and the bacteria incubated on ice for 30 minutes, before they
were heat-shocked at 42°C for 45 seconds and placed back on ice for 2 minutes. To
promote bacterial proliferation 250 µl prewarmed NZY+-broth medium were added and the
bacteria incubated at 37°C for 1 hour with shaking at 225-250 rpm. Half of the bacterial
suspension was then plated on LB-Agar plates as described in the next chapter (2.4.2).
2.4.2.
Transformation
To amplify the generated plasmids for endotoxin free transfection in alveolar epithelial cell
lines the plasmids were transformed into E. coli JM-109 by heat-shock.
Bacteria were thawed on ice and 50 µl of the suspension were transferred to a precooled
reaction tube. Two to 5 µl of the ligation were added and the mixture incubated for 10 min
on ice. Bacteria were then heat-shocked for 45-50 seconds at 42°C and immediately
incubated on ice for another 2 minutes. Now, 450 µl SOC-medium were added and the
bacteria incubated for 1 hour at 37°C, until 100 µl were plated on LB-agar plates
containing the appropriate antibiotic (pCMV3A und pE-eYFP: kanamycin; pcDNA3.1:
ampicillin) to select for successfully transformed bacteria.
2.4.3.
Plasmid preparation
The bacterial colonies that were obtained had to be screened for containing the right, nonmodified plasmid. Small liquid cultures were prepared in 15 ml polypropylene tubes by
inoculating 5 ml of LB-medium with bacteria picked directly from the agar-plate with a
pipette tip, incubated over night at 37°C. Colony PCR (see Table 19) was performed to
test for integration of the right insert with the primers used for cloning. The liquid culture
was stored at 4°C to be able to inoculate a large scale bacterial culture and to prepare
glycerol stocks (1:1 [v/v], stored at -80°C) for further amplification. The PCR-products
were electrophoretically analyzed for the right size. Plasmids of promising clones were
isolated with the Qiaprep Spin Miniprep Kit from the small liquid cultures exactly as
instructed by the manufacturer and sent to sequencing for validation (Seqlab, Göttingen,
Germany).
30
Table 19 PCR reaction colony-PCR (20 µl)
Component
volume
H2O
14.4 µl
buffer
2.0 µl
dNTP
0.4 µl
primer forward
0.5 µl
primer revers
0.5 µl
bacterial culture
2.0 µl
Taq DNA Polymerase
0.2 µl
To generate large amounts of plasmids that are needed for transfections 100 ml LBmedium containing the appropriate antibiotic were inoculated with bacterial culture either
from the stored mini-culture or from the glycerol stock and incubated over night at 37°C
with shaking. Plasmids were isolated with the Qiagen Plasmid Maxi-Kit exactly as
instructed by the manufacturer. Plasmid DNA was disolved in 200-300 µl TE-buffer, the
concentration measured with a NanoDrop photometer and stored at -20°C.
31
2.5. Western-Immuno-Blotting
Quantification of protein levels was done using the western-immunoblot technique. Cells
were treated as indicated and washed three times with ice-cold PBS incl. Ca2+ and Mg2+.
After complete removal of PBS cells were lysed with 300 µl of lysis buffer (mRIPA: 50
mM Tris pH 8.0; 150 mM NaCl; 1 % Igepal; 1 % Na+-deoxycholate) with protease
inhibitor cocktail (complete; 40 µl/ml) on ice for 10 min. The cell lysate was collected and
cleared by centrifugation (10,000 rpm/10 min). Protein concentration was determined with
the Quick-Start Bradford-Assay. Equal protein amounts were diluted in 2 x sample-buffer,
denatured under agitation (97 °C, 350 rpm; 7-10 min) and separated electrophoretically in
a 10 % acrylamide-gel using standard techniques. Proteins were then transferred to a
nitrocellulose membrane with a semi-dry transfer chamber (45-60 minutes, Biorad) and
unspecific binding sites blocked with 5 % [m/v] skim milk powder in TBS-T for 1 hour.
After removal of blocking buffer the membranes were incubated in the primary antibodies
at 4°C over night (see Table 20).
Table 20 Antibodies for Western-Blotting
antibody
buffer
dilution
species
company
V5
TBST + 5 % BSA
1:2000
mouse
Invitrogen
E-cadherin
TBST
1:400
rabbit
Santa Cruz
Actin
TBST
1:2000
rabbit
Sigma
GFP
TBST
1:1000
mouse
Roche
HA
TBST
1:1000
mouse
Covance
Myc
TBST
1:2000
mouse
Invitrogen
Nedd4-2
TBST
1:1000
rabbit
Santa Cruz
Flag M2
TBST
1:1000
mouse
Sigma
tGFP
TBST
1:5000
rabbit
Evrogen
The unbound primary antibodies were removed by washing the membrane in TBS-T three
times for 10 minutes. Membranes were then incubated with the appropriate horse-raddish
peroxidase linked secondary antibodies (rabbit anti-mouse IgG 1:5000; goat anti-rabbit
IgG 1:2000). Excess antibody was removed again by washing the membrane as stated
above, drained and incubated with chemiluminescent substrate (SuperSignal West Pico).
The signal was quantified by exposing an autoradiography film (BioMax MR or
Amersham Hyperfilm ECL) that was developed in a Curix 60 developer. Films were
32
scanned with a CanoScan LIDE 90 and densitometry was performed with ImageJ software
(NIH).
In some cases the signal intensity was too low to be detected by the standard protocol. In
these cases the concentration of the secondary antibody was reduced to 1:50,000-100,000
and the membrane incubated with the SuperSignal West Femto substrate to obtain maximal
detection sensitivity.
2.6. Biotin-Streptavidin-Pulldown
Discrimination between the total cellular amount of ENaC and the functional fraction
located in the plasma-membrane was carried out with biotin-streptavidin-pulldown. The
underlying principle is the conjugation of a modified, membrane impermeable biotin to
lysines located in the extracellular domains of membrane proteins and the subsequent
pulldown with immobilized streptavidin that binds to biotin with high affinity (Gottardi et
al., 1995).
Briefly, cells were rinsed with PBS incl. Ca2+ and Mg2+ and incubated on ice with EZ-Link
Sulpho-NHS-LC-biotin-solution (1 mg/ml in PBS) for 20 min. To quench unbound biotin
cells were washed three times for 10 minutes with PBS containing 100 mM glycine. After
a final washing with PBS cells were lysed as described in chapter 2.5. For each experiment
equal amounts of protein (150-1000 µg) were incubated with 60-100 µl streptavidinagarosebeads at 4 °C over night in a rotator with the volume adjusted with mRIPA-buffer
to obtain equal protein concentrations.
The following day the beads were washed with the solutions indicated below to eliminate
unbound proteins and proteins that were bound unspecifically. Washing solutions were
applied, the tube inverted a couple of times, beads collected at the bottom of the tube by
centrifugation in a mini centrifuge and the supernatant aspirated almost completely after
each step.
1 x solution A: 150 mM NaCl, 50 mM Tris pH 7.4, 5 mM EDTA
2 x solution B: 500 mM NaCl, 50 mM Tris pH 7.4, 5 mM EDTA
3 x solution C: 500 mM NaCl, 20 mM Tris pH 7.4, 0.2 % BSA [m/v]
1 x solution Tris: 10 mM Tris, pH 7.4
Finally the last washing solution was aspirated completely, sample buffer added and the
samples were denatured at 99°C for 8-10 minutes. Analysis was performed as described
above (chapter 2.5). To prove that changes were not caused by varying transfection
33
efficiencies, total cell lysate of every sample was subjected to western blotting without
pulldown.
2.7. Pulse-chase-experiments
To determine the stability of ENaC subunits located membrane, cells were biotinylated as
stated above. After quenching the unreacted biotin with glycine cells were not yet lysed.
Instead, preequilibrated medium was applied and cells put back in the cell culture
incubator for up to 6 hours. After different time points the cells were washed with PBS,
lysed with m-RIPA buffer supplemented with MG-132 (10 µM) and processed as
described in chapter 2.6.
2.8. Immunoprecipitation of detergent insoluble fraction of ENaC
The cell surface expression and composition of the ENaC-complexes are strongly
dependent on the celltype and the underlying culture conditions. Especially for kidney cell
lines it has been reported, that the subunits aggregate to complexes that are insoluble in
several non-ionic detergents (Prince and Welsh, 1998). To test whether this also applies to
alveolar epithelial cell lines the detergent soluble and insoluble fractions were analysed.
A549 and H441 cells were transfected and cultured for 24-48 h in the presence of
amiloride (10 µM) to prevent cell swelling when coexpressing all three ENaC-subunits.
Next, cells were rinsed three times with PBS containing Ca2+ and Mg2+ before cells were
lysed in Tris-buffered saline (TBS) containing 1% Triton X-100 [v/v] and protease
inhibitors (complete, 40 µl/ml). The insoluble fraction was pelleted by centrifugation
16,000 g for 10 minutes at 4 °C. Supernatant was collected in another tube and the pellet
lysed in 100 µl of solubilization-buffer (50 mM Tris (pH 7.4); 2 % SDS [w/v]; 1 % bmercaptoethanol [v/v]; 1 mM EDTA) at 90°C for 5 min. After heating the samples were
diluted in 1 ml TBS containing 1 % Triton X-100. ENaC subunits were
immunoprecipitated from both fractions with subunit specific antibodies (FLAG M2, 0.5
µg/reaction; GFP, 2 µl/reaction) and 100 µl protein A/G beads at 4°C over night.
Samples were finally washed three times with TBS incl. 1 % Triton X-100, denatured in
sample buffer and further processed as described in chapter 2.5.
34
2.9. Statistical analysis and graphical illustration
Data are presented as mean ± SEM, if not described otherwise. Statistical comparison
between two groups was done using an unpaired Student`s t-test. Multiple data sets were
compared by ANOVA and subsequent post hoc analysis. GraphPad prism 6 (GraphPad
software, San Diego, CA) was used for the analysis and data presentation. Data obtained
from Ussing chamber experiments were visualized with FreeHand 10.
35
2.10. Materials
Table 21 Electronic devices
4D-Nucleofector System
Lonza, Köln, Germany
BioPhotometer
Biorad, München, Germany
Developer Curix 60
Agfa, Mortsel, Belgium
Electrophoresis system
Galaxy MiniStar
VWR, Bruchsal, Germany
Hera Cell 150 incubator
Thermo Scientific, Dreieich
Heraeus Fresco 17 thermo centrifuge
Thermo Scientific, Dreieich
KNF Laboport Pumpe
KNF Freiburg, Germany
Magnetstirrer MR 3002
Heidolph, Schwabach, Germany
Mettler H20T precicion scale
Mettler Toledo, Gießen, Germany
Milli-Q water purification
Millipore, Schwalbach, Germany
Mini-PROTEAN Tetra Cell
Msc-Advantage
Thermo Scientific, Dreieich, Germany
NanoDrop (ND-1000)
Kisker-Biotech, Steinfurt
Neubauer counting chamber
Labor Optik, Friedrichsdorf, Germany
PB303 DeltaRange scale
Mettler Toledo, Gießen, Germany
pH-Meter 766 Calimatic
Knick, Berlin, Germany
pipettes
Gilson,
Limburg-Offheim/Biohit,
Rosbach,
Germany
Pipetus
Hirschmann, Eberstadt, Germany
Polymax 1040 Orbitalshaker
Heidolph, Schwabach, Germany
PowerPac Basic
Stratagene MX 3000P
Stratagene, Waldbronn, Germany
Thermocycler Biometra T Personal
Biometra GmbH, Göttingen, Germany
Thermomix ME waterbath
B.Braun Biotech, Melsungen, Germany
Thermomoxer comfort
Eppendorf, Hamburg, Germany
Transblot SD Semi-Dry Transfer Cell
VV3 vortex
VWR, Darmstadt, Germany
Table 22 Reagents
b-mercaptoethanol
Sigma-Aldrich, Steinheim, Germany
2-propanol
Merck, Darmstadt, Germany
7-AAD
Invitrogen, Darmstadt, Germany
A549 cells
LGC, Wesel, Germany
Agarose
Fermentas, St. Leon-Rot, Germany
Amersham Hyperfilm ECL
GE life sciences, Freiburg, Germany
Ammoniumpersulfat (APS)
Promega, Mannheim, Germany
Ampicillin
Sigma-Aldrich, Steinheim Germany
BamHI
BioMax MR autoradiography film
Kodak (distributed Sigma-Aldrich)
Bovine serum albumin (BSA)
Bromphenolblue
Complete Protease Inhibitor
Roche, Basel, Switzerland
Cytotoxicity Detection Kit (LDH)
Roche, Basel, Switzerland (#11644793001)
Dexamethasone
DMEM 1.5 g/l glucose, stable L-glutamine
PAA, Cölbe, Germany
DMEM 4.5 g/l glucose, stable L-glutamine
DNAse/RNAse free water
Gibco, Darmstadt, Germany
2+
2+
DPBS with Ca and Mg
2+
2+
DPBS without Ca and Mg
E. coli JM 109
EcoRI
Ethanol 70 %, 96 % and 100 %
Otto Fischer GmbH, Saarbrücken, Germany
Ethylene-diamin-tetraacetic-acid (EDTA)
EZ-linked-sulpho-NHS-LC-biotin
F12 supplement solution
Filter paper
Glycerol
Glycine
Goat anti-rabbit IgG
Cell signaling, Frankfurt am Main, Germany
H441 cells
LGC, Wesel, Germany
HCl
Carl Roth, Karlsruhe, Germany
Igepal
iScript cDNA Synthesis Kit
iTaq Sybr Green Supermix with ROX
ITS
Kanamycin
KCl
36
KH2PO4
LB agar
LB-medium
Lipofectamine 2000
Lipofectamine RNAiMAX
Methanol
Na+-deoxycholate
+
+
Na -Selenit
Na2HPO4
Na3VO4
NaCl
NaHCO3
NaN3
NaOH
Carl Roth, Karlsruhe,Germany
Natriumdodecylsulfat (SDS)
Opti-MEM
P3 Primary Cell 4D-Nucleofector X Kit
pcDNA3.1V5/Hyg
pCMV-HA-C
Clontech, Saint-Germain-en-Laye, France
pCMV-tag 3
Clontech, Saint-Germain-en-Laye, France
Penicillin/Streptomycin-mixture
Pfu Ultra DNA polymerase
Agilent, Böblingen, Germany
Polypropylene reaction tube 14 ml
BD Falcon, Heidelberg, Germany
Primer
Metabion, Martinsried, Germany
Protein A/G agarose beads
Santa Cruz, Heidelberg, Germany
Q5 DNA polymerase
New England Biolabs, Frankfurt am Main,
Na -Pyruvat
Germany
Qiagen Plasmid Maxi-Kit
Qiagen, Hilden, Germany
Quick-Start Bradford solution
QuikChange Site-Directed Mutagenesis Kit
Agilent, Böblingen Germany
Rabbit anti-mouse IgG
RNeasy-Kit
Qiagen, Hilden, Germany
RPMI 1640
SafeSeal PCR reaction tubes 0.5 und 1.5 ml
Sarstedt, Nümbrecht, Germany
SF Cell Line 4D-Nucleofector X Kit
Snapwell permeable supports #3801
Corning, (Sigma-Aldrich, Steinheim, Germany)
SOC Medium
Streptavidin-agarose beads
SuperSignal West Femto substrate
Pierce, Bonn, Germany
37
SuperSignal West Pico substrate
Pierce, Bonn, Germany
SYBR Safe
T4 DNA Ligase
Tetramethylethylendiamin (TEMED)
Transwell supports #353090
BD Falcon, Heidelberg, Germany
Tris Base
Triton-X 100
Trypsin/EDTA
PAN, Aidenbach, Germany
Tween 20
Sigma-Aldrich, Steinheim Germany
Wizard SV Gel and PCR Clean-Up System
38
39
Table 23 Buffers
Running buffer westernblot 1 l
30 g
Tris
144 g
Glycin
100 ml
SDS (10 %)
Adjusted with millipore water to 1 l
Transfer buffer 1 l
2.45 g
Tris
12.20 g
Glycin
20.00 %
Methanol [vol/vol]
Adjusted with millipore water to 1 l
Washing buffer Tris buffered saline TBS-T pH 7.6; 1 l
1.00 ml
Tween 20
2.42
g
Tris
8.00
g
NaCl
Adjusted with millipore water to 1 l, pH corrected to 7.6
Sample buffer 2 x; 50 ml
5 ml
10 x
Tris 1 M; pH 6.8
6.25 ml
Tris 1 M; pH 6.8
20 ml
SDS (10 %)
2.50 ml
SDS (20 %)
10 ml
Glycerol
5.00 ml
Glycerol (99 %)
Bromphenol blue
Adjusted with millipore water to 50 ml
Stripping buffer 25 ml
2.5 ml
Glycin 1 M
22.5 ml
H2O
250 µl
HCl (37 %)
Incubation for 1 h at room temperature with agitation
Blocking buffer
5 g
100 ml
Skim milk powder
Washing buffer
Bromphenol blue
m-RIPA lysis buffer 100 ml
10 ml
Tris 0.5 M; pH 8
3 ml
NaCl 5 M
1 ml
Igepal (NP-40 substitute)
1 g
Na+-Dexycholat
Adjusted with millipore water to 100 ml
Tris acetic acid EDTA buffer (TAE), DNA electrophoresis 1 l
4.8 g
57.0 ml
100.0 ml
Tris
Acetic acid
0.5 M EDTA pH 8
Adjusted with millipore water to 1 l, pH 8
40
Results
41
3. Results
3.1. Functional Ussing chamber measurements
3.1.1. pH changes of the buffer have no influence on alveolar
Na+-transport
To elucidate the impact of hypercapnia on alveolar Na+-transport, Ussing chamber
experiments were performed. This technique measures the electrogenic transport of ions
through flat tissue like bladder, frog lung, and skin or through artificial cell-monolayer.
The electrical current detected (short circuit current (Isc)) is the sum of all ion transport
processes. Applying pharmacological inhibitors specific for individual ion transporters or
channels makes it possible to fractionate the Isc into different elements. Common inhibitors
used to study Na+-transport are amiloride, that blocks epithelial Na+-channels and ouabain,
which inhibits the Na+,K+-ATPase.
Due to the effect of CO2 on the pH of the buffer, all solutions were buffered with Tris to
compensate for changes of pH, when bubbled with CO2-containing gas-mixtures. Protons
respectively acidification have been shown to regulate ENaC (Awayda et al., 2000; Collier
and Snyder, 2009), so the first set of experiments was performed to show that the slight
changes of pH caused by reperfusion of the solutions that were expected in this open
system did not alter the amiloride-sensitive current IAmi. A drop of the pH from 7.4 to 7.2
and an increase from 7.2 to 7.6 did not result in a significant change of IAmi (Figure 4).
Controls received a constant pH buffer and time-matched amiloride applications.
Results
42
Figure 4 Amiloride-sensitive current during pH variations
The amiloride-sensitive current (IAmi) of polarized H441 cell monolayers was measured during different
acidities of the Ringer‘s solution (pH: 7.2; 7.4; 7.6) in voltage clamp mode. Controls were perfused with
Ringer‘s solution with a constant pH of 7.4 and received time-matched amiloride applications (bars show
mean ± SEM; n = 4; *: P < 0.05).
Results
43
3.1.2. Hypercapnia reduces total INa
Due to the complexity of this Ussing chamber-setting with every solution bubbled
separately, hypercapnia could only be applied at one compartment at a time. When
hypercapnic solution was administered at the apical compartment for 20 minutes, a
significant decrease of IAmi was detected (Figure 5) that was absent when administered at
the basolateral compartment (Figure 6).
Figure 5 Apical application of hypercapnic solution
H441 cells were exposed to normocapnic and hypercapnic Ringer‘s solution and the IAmi measured before
and after 20 min (ami: amiloride 10 µM). Hypercapnic solution was administrated only from the apical side
(graphs show mean ± SEM; n = 4; *: P < 0.05).
Figure 6 Basolateral application of hypercapnic solution
H441 cells were exposed to normocapnic and hypercapnic Ringer‘s solution and the IAmi measured before
and after 20 min (ami: amiloride 10 µM). Hypercapnic solution was administrated only from the basolateral
side (graphs show mean ± SEM; n = 4; *: P < 0.05).
Results
44
3.1.3. Membrane-permeabilization experiments
The epithelial Na+-transport is mainly the product of the activity of the Na+,K+-ATPase
located in the basolateral membrane of polarized epithelial cells and the conductance of the
Na+-channels, situated in the apical membrane. To locate the element(s) affected by
hypercapnia, permeabilization studies were performed. To target only the basolateral
membrane, all amiloride-sensitive channels were blocked, the apical membrane
permeabilized by the antimycotic nystatin and the ouabain-sensitive current IOua measured
after 20 min hypercapnia.
Figure 7 Principle of apical and basolateral membrane permeabilization
During apical permeabilization, Na+-channels are inhibited by amiloride and the apical membrane is
permeabilized by nystatin, enabeling to directly measure the activity of the Na+,K+-ATPase, located in the
basolateral membrane. During basolateral membrane permeabilization, the Na+,K+-ATPase is inhibited by
ouabain and the basolateral membrane permeabilized by nystatin. Since the Na+,K+-ATPase usually creates
the Na+-gradient, but is now deactivated, an artificial Na+-gradient has to be generated by the investigator.
During apical permeabilization a large decrease in IOua was detected indicating a strong
inhibition of the Na+,K+-ATPase during hypercapnia (Figure 8). This is in line with
previous findings (Briva et al., 2007; Vadász et al., 2008, 2012).
Results
45
Figure 8 Ouabain-sensitive Na+-transport during short-term hypercapnia.
Amiloride-sensitive Na+-channels were blocked using amiloride and the apical membrane was permeabilized
with nystatin. Next, control or hypercapnic solution was applied for 20 min and the ouabain-sensitive current
IOua measured (graphs show mean ± SEM; n = 4; *: P < 0.05).
Permeabilization of the basolateral membrane is more complex compared to
permeabilization of the apical membrane. The Na+,K+-ATPase is inhibited by ouabain and
the basolateral membrane permeabilized by nystatin. Since the Na+,K+-ATPase creates the
driving force for Na+ to enter the cells, a Na+-gradient had to be generated artificially. To
prevent a falsification of the current by Cl--ions and to stabilize the electrogenic gradients,
Cl- -ions were substituted with the also negatively charged gluconate. The resulting IAmi, in
the control as well as in the hypercapnia treated cells, was extremely small (Figure 9). No
significant changes between control and hypercapnia-treated cells were detected, but due to
the small current, an effect of CO2 can not be excluded.
Results
46
Figure 9 Amiloride-sensitive Na+-transport during short-term hypercapnia
Ouabain-sensitive Na+-transporter were inhibited with ouabain (1 mM) and the basolateral membrane
permeabilized with nystatin (150 µM) and an artificial Na+-gradient was generated. 20 min later, control or
hypercapnic solution was also applied for 20 min and the amiloride-sensitive current measured (graphs show
mean ± SEM; n = 4; *: P < 0.05).
3.1.4. Long-term
hypercapnia
affects
the
apical
Na+-
conductance
To eliminate possible side-effects of the combination of additional Tris-base, artificial Na+gradient and Cl--substitution and to elucidate the effect of chronic hypercapnia, H441 cells
were incubated in Normocapnia- or Hypercapnia-medium for 24 h and the
permeabilization was repeated with the buffers published previously (Ramminger et al.,
2004; Woollhead et al., 2005) without any buffering and CO2. Interestingly, the IAmi of
cells that were cultured in hypercapnic conditions was significantly reduced compared to
control cells, even after approximately 30 min without CO2 (Figure 10).
A reduction of the IAmi can either be caused by a reduced open-probability (Po) or a
reduced number of channels in the membrane (N). Since the effect of 24 h hypercapnia
was so stable, a change of Po alone is most unlikely. Thus the endocytic pathway was
targeted to investigate a contribution of the change of retrieval of channels from the
membrane. Endocytosis of ENaC has been reported to be mediated by the 5' adenosine
monophosphate-activated protein kinase (AMPK). Attempts to use the AMPK-inhibitor
compound C or the proteasome-inhibitor MG-132, that is also commonly used to block
endocytosis of transmembrane proteins (Gentzsch et al., 2010; Malik et al., 2006) prior to
subjecting the cells to hypercapnia resulted in significant cell death, probably because of
the long incubation times of more than 24 hours.
Results
47
Figure 10 Effect of 24 h CO2 on Na+-conductance
H441 cells were incubated in normo- and hypercapnic conditions for 24 h. Apical permeabilization was
performed immediately and the amiloride-sensitive current was determined (mean ± SEM; n = 3; *: P <
0.05).
Results
48
3.2. rtPCR: Transcription levels of ENaC during hypercapnia
To rule out a regulation of ENaC-subunits on the transcriptional level H441 cells were
exposed to normocapnia and hypercapnia and mRNA was isolated after 6 or 24 hours
(Figure 11). Real-time rt-PCR revealed no change in transcription after 6 hours for all
human ENaC subunits. After 24 hours only the expression of the γ-subunit was
significantly decreased, although probably not to a biologically relevant extent (Figure
12).
Figure 11 Transcription levels of ENaC subunits after 6 h CO2
Native H441 cells were exposed to control conditions (40 mm CO2) or hypercapnia (110 mm CO2), pH 7.4
for 6 h prior to mRNA isolation and subsequent rtPCR (Graphs represent mean, whiskers mark the 5-95
percentile (n = 3) no significant differences were detected).
Results
49
Figure 12 Transcription levels of ENaC subunits after 24 h CO2
Native H441 cells were exposed to control-conditions (40 mm CO2) or hypercapnia (110 mm CO2), pH 7.4
for 24 h prior to mRNA isolation and subsequent rtPCR (Graphs represent mean, whiskers mark the 5-95
percentile; n = 3; unpaired Student’s t-test compared to control; *: P < 0.05).
Results
50
3.3. Expression cloning of modified human ENaC constructs
3.3.1. β-ENaC
Since a pharmacological manipulation of H441 cells during 24 hours hypercapnia was not
feasible in the Ussing chamber setting, a different model had to be established. Mall et al.
reported that overexpression of β-ENaC is sufficient to increase the ENaC-complex
membrane abundance and to cause a cystic-fibrosis-like phenotype in mice (Mall et al.,
2004). Thus, a modified β-ENaC was cloned and overexpressed in alveolar epithelial cells
to enhance surface abundance of all ENaC-subunits. To improve the antibody recognition
of the overexpressed β-ENaC construct, the epitope-tag V5 was added to the C-terminus
(Figure 13).
b-ENaC
96 kDa
V5
95 kDa + 1 kDa V5
Figure 13 β-subunit of the human epithelial Na+-channel was cloned from human lung tissue
The coding region corresponds to the NCBI Reference Sequence NM_000336.2. The epitope-tag V5 was
added at the C-terminus, resulting in a predicted size of 96 kDa.
Results
51
3.3.2. Cell surface abundance of β-ENaC-V5
To assess whether experimental difficulties using the buffered Ussing chamber solution
rendered the effect of CO2 on ENaC invisible, the newly generated β-ENaC-V5 construct
was transfected in A549 cells and the cell surface abundance and total cellular content was
investigated by cell surface biotinylation. After one hour of hypercapnia no changes of the
surface or total ENaC fraction were detected (Figure 14).
A
B
β-ENaC-V5
β-ENaC-V5
E-Cadherin
E-Cadherin
Figure 14 Short-term effect of CO2 on β-ENaC-V5
A549 cells were transiently transfected with β-ENaC-V5. 24 h later the cells were subjected to normocapnia
or hypercapnia for 1 hour and the β-ENaC-V5-levels were determined. (A) Biotin-streptavidin pulldown was
performed to assess cell surface abundance of β-ENaC-V5. (B) Whole cell lysate of the same samples was
blotted to determine total β-ENaC-V5 content (n = 4).
Results
52
In the functional studies a significant decrease of the INa was detected (Figure 10).
Consequently, cell surface expression of β-ENaC-V5 was also investigated after 24 h
hypercapnia. In line with the functional studies a marked decrease of the cell surface
abundance could be observed that was not caused by generally lower total ENaC content
(Figure 15). Pharmacological intervention using the endocytosis and proteasome inhibitor
MG-132, the lysosome inhibitor chloroquine or the AMPK-inhibitor Compound C 2 hours
before and during the 24 h hypercapnia incubation resulted again in significant cell death.
B
1.5
1.5
0.5
0.0
0.0
ro
l
O
co
nt
C
co
nt
β-ENaC-V5
β-ENaC-V5
E-Cadherin
E-Cadherin
O
0.5
2
1.0
ro
l
1.0
2
ns.
*
C
A
Figure 15 Long-term effect of CO2 on β-ENaC-V5
A549 cells were transiently transfected with β-ENaC-V5. Four hours later, cells were subjected to
normocapnic (control) conditions or hypercapnic (CO2) for 24 h. (A) Biotin-streptavidin pulldown was
performed to assess cell surface abundance of β-ENaC-V5. (B) Whole cell lysate of the same samples was
blotted to determine total β-ENaC-V5 content (n = 5; *: P < 0.05).
Results
53
3.3.3. Expression cloning of human α- and γ-ENaC
Against the above mentioned evidence from the literature (chapter 1.3) no acute regulation
of β-ENaC was observed. A possible explanation for that could be a different processing of
individually expressed ENaC-subunits compared to a system in which all three subunits are
expressed simultaneously, as reported for other cell types (Hughey et al., 2003). Thus, αand γ-ENaC constructs were generated. Since both subunits undergo proteolytic processing
during maturation, each subunit was tagged individually at the N-, as well as the Cterminus with different epitope tags (Figure 16) to provide a powerful tool for studying all
aspects of post-translational ENaC-regulation, including but not limited to ubiquitination,
phosphorylation and binding to the E3-ubiquitin ligase Nedd4-2.
eYFP
α-ENaC
b-ENaC
γ-ENaC
118 kDa
96 kDa
97 kDa
Flag
30 kDa + 27 kDa eYFP
V5
95 kDa + 1 kDa V5
60 kDa + 1 kDa Flag
c-Myc
HA
20 kDa + 1 kDa c-Myc
75 kDa + 1 kDa HA
Figure 16 Overview about all ENaC-plasmids that were generated as part of this study
Expected sizes are given of the full length (top) as well as of the mature fragments (bottom) with the genetic
modifications. Modified from R. Hughey (Hughey et al., 2003)
Results
54
MW
150 kDa à
100 kDa à
75 kDa à
50 kDa à
35 kDa à
DNA-amount
2
4
2
4
[µg/transfection]
clone 9
clone 9.2.1
Figure 17 Expression pattern of α-ENaC before and after site-directed mutagenesis
The α-ENaC clone number 9 contained two single nucleotide polymorphisms (A334T, T663A), compared to
the reference sequence NM_001038 (genebank, NIH), which were corrected using site-directed mutagenesis.
The modified clone 9.2.1 contains a coding region identical to the reference sequence and does not exhibit
alternative cleavage pattern when expressed individually in A549 cells (antibody used: anti-GFP, Roche).
α, β and γ-ENaC could be expressed in A549 cells. The membrane abundance however
was very variable and in many cases too low to be detected, even with the most sensitive,
non-radioactive detection methods available for western-blotting. Further, cotransfection
with plasmid amounts in the sublethal range resulted in only very low expression levels of
α and γ ENaC.
Trafficking of fully assembled ENaC-complexes to the cell membrane that are insoluble in
the generally used non-ionic detergent-containing lysisbuffers were reported for different
types of kidney cells (Prince and Welsh, 1998). Additionally a localization of ENaC in
lipid rafts has been described in the mouse and frog kidney cells, also rendering it insoluble
in the above mentioned buffers (Hill et al., 2007). An experiment was designed to evaluate,
whether this was also the case for A549 cells, which exhibit formation of lipid rafts (Song
et al., 2007). α-ENaC alone and α, β and γ-ENaC together were transfected in A549 cells
and the abundance of α-ENaC in the soluble fraction was compared to the insoluble
fraction.
Results
55
As can be seen in Figure 18 α-ENaC was expressed when transfected alone and together
with β and γ (A: lanes 2a, 3a; whole cell lysate, Triton X-100 soluble). An
immunoprecipitation was performed to compare the abundance of α-ENaC in the Triton X100 soluble (b) and insoluble fraction (c) of the same cell lysate. Only faint bands were
detected in the insoluble fraction (lane 2c), suggesting ENaC not to be present in lipid rafts
in A549 cells. As a positive control for succesful immunoprecipitation also the soluble
fractions were subjected to immunoprecipitation (b), which led to a distinct pull down (A
and B; lanes 2b, 3b) that was detectable even after a brief exposure.
A MW
long exposure
IP: anti GFP; IB: anti GFP
150 kDa à
100 kDa à
75 kDa à
50 kDa à
35 kDa à
1a
B MW
2a
3a 1b
2b
3b
1c
2c
3c
short exposure
IP: anti GFP; IB: anti GFP
150 kDa à
100 kDa à
75 kDa à
50 kDa à
35 kDa à
1a
2a
3a
1b
2b 3b
1c
2c
3c
Figure 18 ENaC localisation in soluble fraction and in lipid rafts in A549 cells.
Cells were transfected without plasmid (1), α-ENaC-YFP alone (2) or α-ENaC-YFP, β-ENaC-V5, γ-ENaCHA combined (3) and lysed in TBS containing 1% Triton X-100. The insoluble pellet was boiled in
solubilization buffer containing SDS and β-mercaptoethanol and diluted in TBS containing 1% Triton X-100.
Total cell lysate is presented in the first three lanes (a, 50 µg protein). Immunopreciptation was performed
either from the Triton X-100- (b, ~ 600 µg protein ), or the SDS-soluble fraction of the same amount of cells
(c). Two different exposure times are shown to focus either on the total cell lysate (A) or the
immunoprecipitated fraction (B). Depicted is a representative of three independent experiments.
Results
56
Protein-turnover especially of ENaC is significantly influenced by the system used and the
culture conditions. Before using the system to study the effect of CO2, the baseline stability
of cell surface α-ENaC was determined by pulse-chase experiments. For that, all
membrane proteins are labeled with the membrane impermeable EZ-linked-sulpho-NHSLC-biotin and the cells incubated again in preequilibrated medium for the duration
indicated. After that the cells were lysed and remaining α-ENaC precipitated with
streptavidin-agarose beads. A half-life of α-ENaC, inserted in the plasma-membrane of
remaining surface -ENaC [A.U.]
about 2 h was measured.
α-ENaC-YFP
(surface)
E-cadherin
(surface)
Figure 19 Stability of cell surface α-ENaC-YFP transfected in A549 cells
All proteins located in the plasma-membrane were labeled with biotin. The cells were then covered with
preequilibrated medium and returned to the cell culture incubator. After the indicated time-points, cells were
harvested, subjected to biotin-streptavidin pulldown and the remaining fraction of labeled α-ENaC-YFP was
determined by western-blotting (means ± SEM, n = 3).
If CO2 was a stimulus for ENaC to be retrieved from the plasma-membrane, decreased
stability of the membrane fraction was to be expected. To adress this, membrane-proteins
were biotinylated as described above, but the cells incubated in normocapnia or
hypercapnia medium for 2 and 4 hours (Figure 20).
Results
57
Observed was a slightly but not significantly decreased stability of α-ENaC during
remaining surface a-ENaC [A.U.]
hypercapnia (Figure 20).
α-ENaC-YFP
(surface)
pulse chase a -ENaC
1.5
control
CO2
1.0
0.5
0.0
0
1
2
3
4
time [h]
0h
N 2h
N 4h
H 2h
H 4h
E-cadherin
(surface)
Figure 20 Stability of surface α-ENaC during normocapnia and hypercapnia
All membrane proteins were labeled with biotin. The cells were then covered with preequilibrated
normocapnia (N) or hypercapnia (H) medium and returned to the cell culture incubator. After the indicated
time-points, cells were harvested, subjected to biotin-streptavidin pulldown and the remaining fraction of
labeled α-ENaC-YFP was determined by western-blotting (mean ± SEM, n = 3).
Degradation of ENaC is catalyzed by the E3-ubiquitin ligase Nedd4-2 (Itani et al., 2009;
Kabra et al., 2008; Malik et al., 2005), and therefore it is anticipated, that its genetic
inhibition should increase the total levels of ENaC and prevent the accelerated degradation
during hypercapnia.
Results
58
As depicted in Figure 21, the genetic inactivation of NEDD4-2 was highly effective.
NEDD4-2
Vinculin
control
siRNA
Figure 21 Nedd4-2 dependent cell surface expression of α-ENaC-YFP
(A) Nedd4-2 was efficiently downregulated by siRNA transfection (representative blot of 5 experiments).
The faster degradation of α-ENaC-YFP during hypercapnia was completely abolished
when Nedd4-2 was silenced. Still, degradation of ENaC located in the plasma membrane
was observed and the rate of degradation did not differ from cells that were expressing
Nedd4-2 (Figure 22) . This finding did not correspond to the efficiency of the knockdown,
which was reproducibly highly efficient.
Results
59
B
n.s.
2.0
total -ENaC [A.U.]
1.5
n.s.
1.0
0.5
1.5
1.0
0.5
4
h
O
C
tr
o
co
n
2
l4
h
0
h
2
O
C
co
nt
ro
4
h
l4
h
h
0.0
0.0
0
remaining surface
-ENaC [A.U.]
A
α-ENaC-YFP
(surface)
E-cadherin
(surface)
Figure 22 CO2 dependent stability of α-ENaC-YFP during silencing of Nedd4-2.
The E3-ligase Nedd4-2 was silenced by siRNA transfection. The stability of α-ENaC-YFP was then
compared after 4 hours of normocapnia or hypercapnia in the cell surface (A) and in the whole cell lysate (B)
(mean ± SEM, n = 3).
3.3.1. Transfection of H441 cells
Compared to A549 cells, H441 cells are more widely used for studying Na+-transport
(Albert et al., 2008; Ramminger et al., 2004). Transfection of H441 cells with a new highend 4D-Nucleofector-system was established to circumvent the poor antibodies raised
against the endogenous ENaC subunits. The process called Nucleofection is based on
electroporation and provides an elegant and fast technique for delivering nucleic acids
directly into the nucleus.
After optimization of the Nucleofection H441 cells could efficiently be transfected (
Figure 23). A mean transfection efficiency with the GFP control-plasmid pMaxGFP of ~
60 % was achieved (Figure 24).
Results
100 µm
100 µm
100 µm
100 µm
0 µ pMaxGFP
60
4 µg pMaxGFP
Figure 23 GFP expression of transfected H441 cells
H441 cells (1 x 106 cells/reaction) were nucleofected with and without 4 µg pMaxGFP. Depicted is a typical
of seven independent experiments.
Figure 24 Transfection efficiency of H441 cells achieved by nucleofection
H441 cells (1 x 106 cells/reaction) were nucleofected with 4 µg pMaxGFP plasmid and the efficiency was
determined by flow cytometry gating for GFP-positive cells (mean ± SEM; n = 3; ****: P < 0.0001 ).
Results
61
Figure 25 Viability of H441 cells after Nucleofection
H441 cells (1 x 106 cells/reaction) were nucleofected with 4 µg pMaxGFP plasmid, stained with the dye 7AAD and the viability was determined by flow cytometry (mean ± SEM, n = 3).
Cotransfection of α, β and γ-ENaC was also succesful, as shown in Figure 26. Every
subunit was detected in the same cell lysate using the subunit specific antibodies (α: GPF,
β: V5, γ: HA). The transfection was transient, with fusion proteins being detectable after
24 hours but with a marked decrease in whole cell protein levels already after 48 hours.
MW
b-ENaC V5
α-ENaC YFP
γ-ENaC HA
150 kDa à
100 kDa à
75 kDa à
50 kDa à
35 kDa à
c
t
24 h
c
t
48 h
c
t
24 h
c
t
48 h
c
t
24 h
c
t
48 h
Figure 26 Cotransfection of α β and γ-ENaC in H441 cells
H441 cells were cotransfected without DNA (c) and with α-ENaC-YFP, β-ENaC-V5, γ-ENaC-HA (t) and
lysed 24 h respectively 48 h later and analysed by western immunoblotting using antibodies against the
respective epitope tags.
Results
62
However, detection of α and γ-ENaC in the cell membrane remained suboptimal. To
optimize the cell surface expression of this two subunits, α and γ-ENaC constructs, both
eGFP-tagged for recognition with the same antibody (Figure 27), were transfected in
H441 cells and analyzed (Figure 28). Surface expression of α and γ-ENaC was evanescent,
considering that the pulldown was performed from 1.8 mg total protein (surface) and the
amount of total cell lysate that was loaded (50 µg / lane; (total)). The chemiluminescent
signal was developed with the Supersignal West Femto substrate for maximal sensitivity.
Treatment of the cells 5 days prior transfection and after transfection with dexamethasone,
as well as aspiration of the apical liquid to expose the cells to air, did not increase the
expression levels within the time-course of plasmid-expression (24 hours, data not shown).
α-ENaC
γ-ENaC
117 kDa
122 kDa
30 kDa
90 kDa tGFP
GFP
20 kDa
102 kDa GFP
GFP
Figure 27 Scheme of the commercially available tGFP-tagged α- and γ-ENaC constructs (Origene)
used in this study
Predicted sizes are given of full length forms (top) and after proteolytical processing (bottom).
Results
MW
63
ǀ
membrane
whole cell
150 kDa à
100 kDa à
75 kDa à
α-tGFP
+
+
α-eYFP/Flag
+
+
b-V5
+
γ-tGFP
+
γ-HA
+
+
+
+
+
+
+
+
+
+
+
+
Figure 28 Expression pattern of α and γ-ENaC in the cell-surface of H441 cells
Combinations of different ENaC-subunits were cotransfected in H441 cells and the cell membrane
abundance compared to the whole cell content of α and γ-ENaC (anti-tGFP). Shown is a biotin-streptavidinpulldown of 1800 µg total protein (membrane) and 50 µg whole cell lysate.
Results
64
3.3.2. Transfection of primary rat alveolar epithelial cells
Primary cells are in many ways superior to immortilized or tumor cell lines and represent a
more physiologic system to study Na+-transport. However, immunological detection of
ENaC has always been difficult in primary cells. Another caveat is that genetic
manipulation of primary cells by gene silencing and overexpression was usually associated
with virus-mediated gene transfer, resulting in non-conventional experimental procedures
that were limited by safety-regulations and complex and labour-intensive virus-production.
As a proof of principle, ATII cells were transfected with Lipofectamine 2000 basically as
described before (2.1.2). The amount of DNA per transfection was increased to achieve the
same ratio of DNA to cells, as with Nucleofection. Subsequently, the amount of
Lipofectamine 2000 was adjusted. The transfection resulted in only a few transfected cells,
as depicted in Figure 29, confirming the dogma, that ATII cells can not efficiently be
transfected by non-viral techniques.
Figure 29 Lipofection of ATII cells
ATII cells were plated on 60 mm cell culture plates and allowed to recover for one day. They were then
transfected with 15 µl Lipofectamine 2000 without or with 5 µg of the vector pMaxGFP. GFP expression
was assessed 2 days later by fluorescence microscopy (n = 3).
Nucleofection is a transfection method, specifically designed for primary cells and other
cells that are hard to transfect. This also applies to ATII cells, therefore a protocol was
created to transfect them. For the first time a safe, reliable and efficient transfection
procedure based on electroporation was established as part of this thesis to genetically
manipulate primary ATII cells. Nucleofection proved to be dose-dependent, as depicted in
Results
65
Figure 30 and Figure 31. The transfection efficiencies for 3, 5 and 8 µg pMaxGFP were
32.4 ± 0.5, 39.6 ± 1.2 and 48 ± 0.7 % respectively (Figure 31).
Figure 30 Nucleofection of primary rat alveolar epithelial type II cells.
Freshly isolated ATII cells were allowed to recover over night on a cell culture dish. The next day 3.5 x 106
cells were transfected with up to 8 µg pMaxGFP plasmid. The negative control was pulsed as all other cells,
but without plasmid DNA and results were visualized 48 hours after transfection.
Figure 31 Transfection efficiency of ATII cells
Freshly isolated rat alveolar epithelial type II cells were allowed to recover on culture dishes over night,
transfected with pMaxGFP (3-8 µg/reaction) and plated onto permeable supports at a density of 3.5 x 106 per
Results
66
filter. Efficiency was assessed two days after transfection. (A) Scatter plot of GFP expression measured by
FACS analysis. (B) Transfection efficiency of AT II cells transfected with the indicated DNA-amounts (bars
indicate mean ± SEM; n = 2-3; ****: P < 0.0001 ).
Cells transfected with 8 µg DNA took longer to attach to the transwell-membrane after 24
h. To rule out early cell death due to the transfection procedure lactate-dehydrogenase
(LDH) release was measured 4 and 24 h after transfection. After 4 h a strong release of
LDH was detected only in cells that received the electrical pulse, which returned to
baseline 20 h later. This LDH release is the result of the formation of pores in the cell
membrane, which are the reason for delivery of nucleic acids by Nucleofection. At the later
timepoint no significant LDH was measured (Figure 32). Counting the number of cells
that remained on the permeable support 48 h after Nucleofection showed no significant
differences (Figure 33).
However, viability was not significantly decreased 48 h after transfection compared to
control cells as assessed by flow cytometry (Figure 34) and the cells formed electrically
tight monolayers with a resistance of about 1200 Ω * cm2, that did not differ from each
LDH release (% of max.)
other (Figure 35).
*
*
no
no
5 µg
8 µg
DNA
pulse
DNA
DNA
Figure 32 Evaluation of early cytotoxicity in transfected ATII cells
ATII cells were transfected as indicated and plated onto permeable supports. Four (patterned bars) and 24 h
(full bars) after transfection medium was removed, cleared from cells and subjected to a lactatedehydrogenase assay to screen for cell damage (mean ± SEM; n = 3; *: P < 0.05).
Results
67
cell number (rel. to no pulse)
ns
no
no
5 µg
8 µg
pulse
DNA
DNA
DNA
Figure 33 Cell number 48 h after Nucleofection
ATII cells were transfected as indicated, maintained for 48 h, trypsinized and the number of attached cells
counted for each experimental condition (mean ± SEM; n = 3).
Figure 34 Viability of transfected ATII cells.
Rat alveolar epithelial type II cells were transfected with pMaxGFP, plated onto permeable supports at a
density of 3.5 x 106 per filter. Control cells were sham transfected without the vector pMaxGFP but
otherwise received the same treatment as cells receiving the GFP vector pMaxGFP (3-8 µg / reaction). Two
days after transfection cells were trypsinized, stained with 7-AAD and subjected to flow cytometry. (A)
Scatter plot of flow cytometry showing live cell population (inside the box). (B) Viability of transfected cells
(bars indicate mean ± SEM; n = 3-4).
Results
68
TEER (% no DNA)
ns
no
DNA
no
pulse
5 µg
DNA
8 µg
DNA
Figure 35 Electrical resistance 48 h after Nucleofection
Two days after Nucleofection the electrical resistance of the ATII cell monolayer was measured with an
volt ohm meter (bars indicate mean ± SEM; n = 3).
Results
69
3.3.3. ENaC transfection in ATII cells
To translate the newly established system to the initial project of studying ENaC and its
regulation during hypercapnia α- and β-ENaC individually and αβγ-ENaC together were
transfected into ATII cells. In the first experiments expression was evaluated two days
after Nucleofection, but no signals were detected (not shown). When the analysis was
carried out already after one day, relatively strong expression of individually transfected αand β-ENaC was detected and faint bands in the cotransfected sample. The low amount of
protein that was available might explain the absence of γ-ENaC (Figure 36). Of note: Due
to the low yield of protein the biotin-streptavidin pulldown was performed from only up to
200 µg protein and only 10-16 µg protein of whole cell lysate was blotted, as opposed to
1.8 mg that were used for the pulldown in H441 cells (chapter 3.3.1).
membrane
ǀ
whole cell lysate
E-cadherin à
α-tGFP
à
b-V5
à
pMaxGFP
α-tGFP
b-V5
γ-tGFP
+
+
+
+
+ +
+
+
+
+ +
+
Figure 36 Pilot experiment for transfection of ENaC subunits in ATII cells
Freshly isolated ATII cells were allowed to recover from isolation for 1 day. They were then transfected
as indicated (equal ratios of subunits, total amount of DNA always 8 µg) and expression of recombinant
ENaC subunits was monitored 24 h after transfection. Pulldown was performed from up to 200 µg protein
and whole cell lysate shows 10 – 16 µg protein.
Discussion
70
4. Discussion
4.1. Hypercapnia modulated regulation of alveolar Na+- transport
The alveolar fluid transport is tightly regulated in the intact lung due to secondary active
Na+-reabsorption creating an osmotic gradient for water to follow passively. The two key
molecules involved in Na+-transport are the Na+,K+-ATPase that actively exchanges Na+
for K+, thus lowering the intracellular Na+-concentration. This drives Na+ from the alveolar
space to passively enter the cells through ENaCs (Matthay et al., 2002).
During different respiratory diseases ventilation is impaired leading to a reduced
elimination of CO2 in the body, a condition called hypercapnia. Hypercapnia can also be a
consequence of lung protective ventilation during ARDS (Hickling et al., 1994).
Despite its immunoregulatory function hypercapnia has been reported to negatively affect
alveolar fluid transport independent of pH. This finding has been reported from rats in
vivo, isolated rat lungs as well as from isolated epithelial cells (Briva et al., 2007). The
underlying mechanism is a CO2 - triggered rapid increase in cytosolic Ca2+. This leads to a
Ca2+-calmodulin dependent kinase kinase β- (CaMKK-β) mediated phosphorylation of the
α-subunit of AMPK. One of the distal elements of this signaling cascade is protein kinaseC-ξ (PKC-ξ) which directly phosphorylates the α-subunit of the Na+,K+-ATPase at serine18, leading to its endocytosis (Briva et al., 2007; Vadász et al., 2008). Phosphorylation of
serine-18 has been shown to induce ubiquitination of the α-subunit of the Na+,K+-ATPase
during hypoxia, so this is likely also the case in hypercapnia (Dada et al., 2007). Activation
of AMPK was also shown to be dependent on extracellular-signal regulated kinase (ERK)
activation.
In the present study, the effect of hypercapnia on Na+-transport in H441 cells has been
tested in Ussing chamber experiments. To rule out any contribution of pH changes, an
initial experiment was performed, showing that variations of pH in the range of 7.2 – 7.6
did not influence the Isc. All other experiments were performed at a pH of 7.4. In line with
the investigations mentioned above, hypercapnia resulted in a pronounced decrease in the
total Na+-transport which was caused by a strong inhibition of the Na+,K+-ATPase activity,
as assessed by membrane permeabilization, supporting the established role of the Na+,K+ATPase in hypercapnia. Interestingly this decrease was only present when hypercapnic
solution was perfused in the apical compartment.
Discussion
71
An explanation could be a distinguished difference in the CO2 permeability of the apical
and basolateral membrane caused for example by different subsets of transmembrane
proteins, in especially gas permeating channels. Aquaporin 1 (AQP1) was demonstrated to
be a physiologically relevant candidate to conduct CO2 (Boron, 2010; Musa-Aziz et al.,
2009; Wang et al., 2007) but its significance as a gas channel is highly controversial. A
study using AQP1 deficient mice did not yield any differences in the CO2 transport rate of
the alveolo-capillary barrier (Fang et al., 2002). Additionally, AQP1 in the lung is
predominantly expressed in endothelial cells (Jiao et al., 2002; King and Agre, 2001; King
et al., 2002), so a potential differential expression pattern of AQP1 in the apical and
basolateral membrane of H441 cells cannot be the reason for the absence of basolateral
CO2 mediated modulation of INa. Other channels that conduct CO2 are AQP4 and AQP5
and AQP 5 is indeed expressed in ATI cells (Verkman et al., 2000). Another possibility
could be an interference of the permeable support with the exposition of the cells to CO2.
These possibilities are currently under investigation in our laboratory.
Interestingly, many elements of the signaling cascade that induces downregulation of the
Na+,K+-ATPase are also known direct or indirect regulators of ENaC function.
Figure 37 CO2 induced signaling cascade leading to endocytosis of the Na+,K+-ATPase
Elements proven to regulate ENaC are highlighted in red.
Discussion
72
ERK has been shown to phosphorylate β- and γ-ENaC thereby mediating interaction with
Nedd4-2 ultimately resulting in endocytosis and reduction of P0 (Shi et al., 2002). Also αENaC expression and additionally alveolar Na+-transport have been shown to be regulated
in an ERK dependent manner (Frank et al., 2003).
PKC mediates liquid regulation in the rat lung (Soukup et al., 2012). Also other groups
report PKC-dependent ENaC regulation (Awayda et al., 1996; Shimkets et al., 1998;
Stockand et al., 2000), but the CO2-activated ξ-isoform has never been directly linked to
ENaC.
JNK on the other hand has been shown to phosphorylate the E-3 ligase Nedd4-2, which is
known to ubiquitinate ENaC, causing its inhibition (Hallows et al., 2010).
AMPK dependent ENaC regulation has been extensively investigated. Activation of
AMPK leads to decreased ENaC function and thus alveolar fluid clearance (Albert et al.,
2008; Carattino et al., 2005; Myerburg et al., 2010; Woollhead et al., 2007). Two
mechanisms seem likely to happen upon activation of AMPK: The classical way of
interaction involves AMPK-dependent association of Nedd4-2 with ENaC subunits that
induces retrieval of the channel from the membrane and thereby limiting Na+-transport
(Bhalla et al., 2006; Carattino et al., 2005). Another way of AMPK-dependent ENaC
regulation is a reduction of single-channel P0 rather than an effect on N as shown in H441
cells (Albert et al., 2008).
Since all these elements are activated during hypercapnia and are associated with ENaC
regulation, the effect of CO2 on ENaC function was investigated as part of the present
study. As mentioned above, total Na+-transport and activity of the Na+,K+-ATPase was
reduced by CO2. Elucidating the effect on the apical Na+-permeability alone revealed no
significant differences in normocapnia- versus hypercapnia-treated cells during up to 20
min.
However, when cells where incubated in normocapnic versus hypercapnic conditions for
24 hours, a marked decrease of the apical Na+-permeability was detected, suggesting an
immediate effect of CO2 on the Na+,K+-ATPase and a delayed effect on ENaC in this
system as postulated previously (Woollhead et al., 2007). Further experiments revealed
that no change of mRNA-levels of all ENaC subunits occurred after 6 and 24 hour,
pointing to a post-translational regulation as opposed to a change in gene-regulation.
Unfortunately a pharmacological intervention with the AMPK-inhibitor Compound C even
in a four fold lower concentration as published by others (Albert et al., 2008) and the
proteasome-inhibitor MG-132 was not successful due to the much longer incubation times,
Discussion
73
so there were no tools left to continue with functional experiments. Efficient transfection of
H441 cells was not yet established so another system had to be used to study the effects of
CO2 on ENaC.
4.2. Molecular mechanism of CO2-regulated ENaC function
The molecular basis of CO2-mediated ENaC regulation was characterized in A549 cells.
These cells are derived from a human alveolar cell carcinoma and contain multilamellar
cytoplasmic inclusion bodies and secrete surfactant similar to primary ATII cells (Lieber et
al., 1976). A549 cells are not ideal for functional alveolar epithelial barrier studies, for
example Ussing chamber experiments, since they exhibit low transepithelial electrical
resistance due to a reduced synthesis of tight-junction proteins such as zona-occludens
protein-1 (ZO-1) (Lehmann et al., 2011). But an extended range of mechanistic
experiments is possible with this system, since genetic manipulation can be done easily.
In the present study A549 cells were used as recipients for epitope-tagged ENaC plasmids,
since endogenous ENaC-proteins are difficult to detect using standard western blot
techniques. In initial suudies, only the human β-ENaC was cloned and modified with a
small epitope V5-tag of the paramyxovirus of simian virus 5 (SV5) for better antibody
recognition. Some evidence from the literature suggests, that regulation of only β-ENaC
might be sufficient for a modulation of ENaC-function: In the human embryonic kidney
cell line (HEK-293) activation of AMPK increased only the interaction of β-ENaC with
Nedd4-2, leading to reduced Na+-transport (Bhalla et al., 2006). Further, overexpression of
β-ENaC alone, but not α or γ in the mouse led to increased fluid reabsorption (Mall et al.,
2004). Thus the rationale of the experiments described now was to produce an ENaC
subunit that could be detected easily and to generally increase the protein levels of all
ENaC subunits in the plasma membrane.
To investigate effects of hypercapnia, transfected cells were exposed to normocapnia and
hypercapnia for 1 and 24 hours and the membrane as well as the whole cell abundance of
β-ENaC was determined. In line with the Ussing chamber experiments no change of βENaC was detected after short term exposure to CO2. After 24 hours of CO2 a marked
decrease of β-ENaC in the cell membrane was observed that was not paralleled by a
decrease of total β-ENaC levels.
Also in line with the Ussing chamber experiments interference with the CO2 induced
signaling cascade using different drugs in β-ENaC expressing A549 cells was not
successful. Overexpression of single ENaC subunits compared to overexpression of the full
Discussion
74
αβγ-ENaC might interfere with posttranscriptional modification and trafficking as reported
by Hughey et al. for Chinese hamster ovary (CHO) and Madin-Darby canine kidney type 1
(MDCK) cells (Hughey et al., 2003). To rule out effects of hypercapnia on the poreforming α- and the regulatory γ-subunit and effects that were limited to the fully functional
ENaC complex, plasmids encoding these proteins modified with individual tags for the Nas well as the C-terminus were generated.
The coding sequence for α-ENaC contained the two single-nucleotide polymorphisms
A334T and T663A that were corrected before starting the actual experiments. Both
polymorphisms are known, but especially T663A was important to correct, since
degradation and activity of both polymorphisms seem to be different. A334T does not
appear to change the physiologic properties of the channel (Tong et al., 2006; Yan et al.,
2006). As demonstrated in the present study both polymorphisms have no effect on the size
and posttranscriptional processing of α-ENaC.
Next, all three ENaC subunits were cotransfected in A549 cells. β-ENaC could be detected
best, but the α- and the γ-subunit were hardly detectable, especially in the membrane. Now,
the ENaC subunits could be recognized by antibodies, but the expression level was
extremely low and in most cases below the detection limit. In the kidney cell lines COS-7
and HEK-293 it has been demonstrated, that ENaC subunits form a complex that is largely
insoluble in conventional detergent-based cell lysis buffers and that ENaC detection was
largely improved, when the detergent-insoluble fraction was denatured in SDS and βmercaptoethanol containing buffer at 90 °C (Prince and Welsh, 1998). Also a trafficking of
ENaC into lipid rafts in kidney cells of the mouse was reported, although the ratio of
insoluble ENaC was smaller in that study compared to detergent-insoluble proteins (Hill et
al., 2007).
In this study, the detergent insoluble fraction of the cell lysate of A549 cells was denatured
as published previously (Prince and Welsh, 1998), but an insoluble complex composed of
αβγ-ENaC could not be detected. Also an incorporation of α-ENaC alone into lipid rafts
that are solubilized by the protocol used could not be shown, indicating that differential
solubility might be a feature of ENaC that is limited to renal cell lines.
Coexpression of all three ENaC-subunits in A549 cells was not succesful. To test whether
α-ENaC alone would be regulated during hypercapnia the half-life of α-ENaC located in
the plasma membrane was determined by pulse chase experiments which revealed a halflife of about two hours. Next, the stability of α-ENaC located in the membrane was
compared during normocapnia and hypercapnia in which a clear trend to reduction in
Discussion
75
hypercapnia, compared to normocapnia, was evident, although no statistical significance
was apparent.
Since the stability of ENaC is described to be markedly affected by its ubiquitin ligase
Nedd4-2 (Kabra et al., 2008; Rotin and Staub, 2012; Staub et al., 2000) a genetic approach
was chosen in which Nedd4-2 was downregulated by siRNA in A549 cells. α-ENaC was
transfected two days later, to match its maximal expression with the time of best
knockdown of Nedd4-2. Baseline degradation seems not to be critically dependent on
Nedd4-2, since about 60 % of α-ENaC located in the membrane was degraded within four
hours independently of the presence of Nedd4-2.
To sum up all experiments involving hypercapnia and A549 cells: β-ENaC is down
regulated after sustained hypercapnia by an unknown posttranslational mechanism,
whereas α-ENaC might already be affected earlier, but definitive evidence is missing.
Nedd4-2 does not seem to play an important role in ENaC degradation in A549 cells,
neither under normal conditions nor during hypercapnia, at least not in the conditions that
were used in this study.
4.3. Transfection of H441 cells – Seeking the right system
After the addition of the 27 kDa eYFP tag, the full size α-ENaC has a predicted size of
about 120 kDa and two fragments of approximately 60 kDa when cleaved. Interestingly,
only the bigger of the two detected bands (~ 100 and 120 kDa) for α-ENaC matched the
predicted size. To complement the experiments that were performed in A549 cells, another
cell line was used for further studies.
An alternative for A549 cells as recipients for genetically modified ENaC constructs are
H441 cells. As mentioned above these cells were used for functional studies of ENaC
activity, but transfection has been difficult and its efficiency in H441 cells has rarely been
published. Low efficiencies of 5 – 10 % were achieved by Polyfect transfection agent
(Lazrak et al., 2009). Virus-mediated gene transfer has been reported to work very well,
since lentiviral transduction reached an efficiency of up to 100 % (Aarbiou et al., 2012).
But virus mediated gene transfer is associated with strict safety regulations and it is very
time and labour intensive.
In the present study, a non-viral protocol was established to transfect H441 cells by
Nucleofection. As shown by fluorescence microscopy and flow cytometry about 60 % of
H441 cells expressed the eGFP encoded in the transfected control plasmid.
Discussion
76
Using the generated ENaC plasmids encoding αβγ-subunits it was possible to transiently
coexpress all three subunits in the same cells. Surprisingly the cleavage pattern of the αsubunit (~ 45 and 130 kDa) was different from the one seen in A549 cells (~ 100 and 120
kDa), although the exact same plasmid was used for transfection. The predicted sizes for
the eYFP-tag containing cleaved and full length forms are ~ 60 and 120 kDa based on the
observations from Hughey and Carattino (Carattino et al., 2006; Hughey et al., 2004), so
the pattern found in H441 cells hints more to a proteolytically activated form of α-ENaC.
Also the cleavage pattern of the γ-subunit was close to the predicted sizes of 76 and 97
kDa, indicating that the posttranslational processing of ENaC in H441 cells was more
likely to function properly compared to A549 cells. However, membrane expression in
H441 cells especially of the α- and γ-subunit was extremely low, close to the detection
limit, although large amounts of proteins were used for the pulldown. Despite extensive
effort on optimization the system could not be fully applied to mechanistic experiments.
Identification of ENaC on the protein level and its interpretation is generally difficult.
Results vary widely dependent on the type of cells that is used and the culture conditions.
And even in the same cell type the sizes for ENaC subunits can be different. Possible
explanations are different states of glycosylation and proteolysis. A brief overview about
endogenous ENaC sizes that have been published is provided in Table 24.
Many investigators use overexpression of hetero- or homologous ENaC subunits, thus
complicating the comparison of different studies. Westernblot results vary from many
nonspecific bands and smears (Bhalla et al., 2006) to a single band for all forms of
cotransfected αβγ-ENaC modified with the same epitope tag (Lee et al., 2009).
Table 24 Reported sizes [kDa] of endogenous ENaC in different cell types (n.d.: not detected)
Cell type
Species
α
β
γ
Publication
ATI/II
Rat
180-200
H441
Human
65 / 67; 90-100
ATII
Rat
60; 90
ATII
Rat
65; 70-75; 97; 150
n.d.
150
(Lazrak et al., 2012)
A6
Xenopus
< 90
90
95
(Eaton et al., 2010)
A6
Xenopus
75; 150; 180
97
95
(Weisz et al., 2000)
A549 cells
Human
100; 120
100
H441
Human
45; 130
100
76; 100
present work
ATII
Rat
120
100
n.d.
present work
(Johnson et al., 2002)
88
(Albert et al., 2008)
(Frank et al., 2003)
present work
Discussion
77
4.4. Novel technique for transfection of primary alveolar epithelial
cells
A549 and H441 cells are both immortalized cell lines that resemble alveolar epithelial
cells, but show clear differences to freshly isolated primary cells (Lieber et al., 1976;
Ramminger et al., 2004). So the most physiologic attempt to study the function of alveolar
epithelia would be to use primary cells.
To combine the generated ENaC constructs with the advantage of primary cells, a novel
technique was established to genetically manipulate primary rat alveolar epithelial type II
(ATII) cells.
ATII cells are hard to transfect. This is basically due to two reasons: Primary alveolar cells
do not proliferate, at least not to a significant extent and contain large amount of
multilamellar bodies.
Only a very small fraction (0.5-1 %) of freshly isolated ATII cells has been reported to be
proliferative (Kalina et al., 1993). Many transfection methods are based on the exposure of
the nuclear machinery to the cytosol during mitosis (Kirton et al., 2013). One example that
was also used in this thesis for A549 cells is lipofection, a lipid based transfection
procedure that works well in rapidly dividing cell lines. This technique incorporates
nucleic acids into liposomes that fuse with the cell membrane, thereby releasing the nucleic
acids into the cytosol (Felgner et al., 1987). As shown in the present thesis the transfection
rate by Lipofection is minimal.
Further, ATII cells contain a large amount of lamellar bodies. Even if siRNA or cDNA can
be delivered to ATII cells, it is trapped and unable to translocate into the nucleus (Friend et
al., 1996).
Two publications suggest that freshly isolated ATII cells take up “naked” siRNA without
any additional treatment (Jain 1999, Jain 2001), but this technique has not been applied and
published any further.
Adenovirus mediated gene transfer, the only efficient way to deliver nucleic acids to ATII
cells, has been reported by several investigators (Berger et al., 2011; Factor et al., 1998;
Roux et al., 2005; Vadász et al., 2012). The transfection efficiency ranges somewhere
around 50 – 60 %. But virus infection is associated with increased safety regulations and
the preparation of the reagents is expensive and time consuming.
During the present study, an innovative transfection method has been established for ATII
cells. The process called Nucleofection is based on electroporation and combines special
nucleofection solutions with distinct electrical pulses to drive nucleic acids directly into the
Discussion
78
nucleus of hard-to-transfect cell lines (Gresch et al., 2004). The best combination is
specific for each cell type and has to be determined empirically.
For optimization purposes an GFP expressing vector was used, so that the successfully
transfected cells could be identified by flow cytometry and fluorescence microscopy. After
optimization the transfection efficiency was approximately 50 %. The determination of the
transepithelial electrical resistance showed no significant reduction of monolayer integrity
of transfected compared to untransfected cells. Immediately after Nucleofection a transient
release of lactate-dehydrogenase was observed in cells that received the electrical pulse.
This LDH release was limited to the transfection procedure and can be explained by the
formation of pores in the cell membrane that enable entry of nucleic acids (Gresch et al.,
2004). LDH release one day after Nucleofection showed no increased cell damage and also
determination of viability two days after Nucleofection was not different from control cells
as assessed by flow cytometry, indicating that the described procedure does not interfere
with cell viability.
Nucleofection of rat ATII cells has already been published before, but the transfection
efficiency for cells harvested from adult animals, as in the present study, was only ~ 13 %.
The authors were using an earlier device and different solutions and electrical pulses
(McCoy et al., 2006). The present study, which has been recently published in part
(Grzesik et al., 2013), demonstrates for the first time highly efficient transfection of
primary rat alveolar epithelial type II cells and thus represents an important advance in
studying physiology of primary alveolar epithelial cells. The non-viral procedure is highly
efficient, non-cytotoxic, fast and since the electrical properties of the plated cells were not
impaired, which indicates that nucleofected ATII cells can be used in a variety of
experimental settings, including electrophysiology.
The newly established Nucleofection protocol was finally used to transfect ATII cells with
ENaC subunits. For convenience, the already described commercially available α- and γENaC constructs (Origene) were used. Preliminary results show that the cell surface
abundance of at least α- and β-ENaC are drastically increased in ATII cells, compared to
H441 cells. Thus, Nucleofection of epitope tagged ENaC-subunits into ATII cells could be
the solution to overcome the poor detectability and low expression levels of ENaC in other
systems.
Summary
79
5. Summary
Acute respiratory distress syndrome is a life threatening condition triggered by a variety of
pulmonary and extrapulmonary causes, that is characterized by pulmonary edema and
subsequently impaired gas exchange. Due to lung protective ventilation strategies, its
treatment is often associated with systemic accumulation of CO2, a condition termed
permissive hypercapnia. Recent studies report a negative effect of CO2 on alveolar fluid
clearance, a process mediated by its two key elements the Na+,K+-ATPase and epithelial
Na+-channels (ENaCs). A reduced activity of the Na+,K+-ATPase during hypercapnia has
already been demonstrated, but regulation of ENaC has never been directly linked to CO2.
Many molecular signaling events that are activated during hypercapnia are known to
regulate ENaC function, so the present study aimed to generate and subsequently apply
techniques to investigate a possible contribution of ENaC to the reduction of alveolar
epithelial fluid transport upon hypercapnia.
ENaC function was studied in H441 cells by Ussing chamber experiments which revealed
no significant regulation during short term hypercapnia, but a clear reduction of ENaC
function during sustained hypercapnia.
To identify the signaling mechanism on the molecular level, epitope-tagged human ENaC
constructs for the α-, β- and γ-subunit were cloned and initially expressed in A549 cells.
Exposition to hypercapnia up to 4 hours did not significantly reduce cell surface expression
of the ENaC-subunits, but after 24 hours, a significant decrease of β-ENaC was observed.
Since the molecular sizes of α- and γ-ENaC expressed in A549 cells were differing from
previously published studies, transfection of ENaC was continued in other cells. H441 cells
are commonly used for ENaC studies, so their transfection was established, yielding an
efficiency of about 60 %. The molecular sizes of transfected ENaC subunits matched the
pattern that was expected, but expression levels were evanescent and too low for further
experiments. Since ENaC detection in these two cell lines remained problematic, a novel
methodology was applied. Since the primary site of ENaC expression in the lung are
epithelial cells, rat primary alveolar epithelial cells type II were used as recipients for
ENaC plasmids. Non-viral transfection of ATII cells has been inefficient in the past, but
during the present study a protocol was generated to efficiently deliver nucleic acids to
exactly this cell type. ENaC expression was largely increased in ATII cells, compared to
the cell lines used, indicating that established system might be extremely useful for further
studies involving ENaC turnover.
Summary
80
Thus, a new and highly relevant, non-viral transfection technique for primary alveolar
epithelial type II cells was established, providing ground-breaking opportunities for future
pulmonary research.
Zusammenfassung
81
6. Zusammenfassung
Das Atemnotsyndrom des Erwachsenen ist eine lebensbedrohliche Erkrankung, ausgelöst
durch eine Reihe von Faktoren, die direkt oder indirekt auf die Lunge einwirken .
Charakteristisch für dieses Syndrom sind pulmonare Ödeme und daraus resultierend ein
eingeschränkter Gasaustausch. Die daher benötigte künstliche Beatmung führt im Zuge
von protektiven Beatmungsstrategien oft zu einer systemischen Anreicherung von CO2
(Hyperkapnie). Einige Studien zeigen, dass erhöhte CO2-Level den Flüssigkeitstransport
der Lunge einschränken. Dieser aktive Prozess wird maßgeblich durch zwei Komponenten,
die
Na+,K+-ATPase
und
epitheliale
Na+-Kanäle
(ENaCs),
kontrolliert.
Eine
Beeinträchtigung der Na+,K+-ATPase durch CO2 gezeigt, für ENaCs ist dies bislang nicht
bekannt. Einige bekannte Regulatoren von ENaCs werden jedoch während Hyperkapnie
aktiviert. Das Ziel der vorliegenden Arbeit war, Methoden zu etablieren und anzuwenden,
die einen möglichen Einfluss von CO2 auf ENaC zeigen.
Funktionelle Versuche wurden an H441-Zellen mit
Ussing-Kammer-Messungen
durchgeführt. Während akuter Hyperkapnie konnte keine signifikante Regulation von
ENaC nachgewiesen werden, jedoch war die ENaC-Funktion bei anhaltender Hyperkapnie
deutlich verringert.
Um die Signalwege auf molekularer Ebene zu untersuchen, wurde die α-, β- und γUntereinheit des humanen ENaC kloniert, genetisch modifiziert und in A549 Zellen
überexprimiert. Nach bis zu vierstündiger Hyperkapnie erfolgte keine Regulation von
ENaC, jedoch wurde nach 24 Stunden eine deutlich verminderte Menge β-ENaC in der
Zellmembran nachgewiesen. Da die Größen von α- und γ-ENaC von den bisher
publizierten abwichen, wurden weitere Versuche in H441 Zellen durchgeführt. Die
Transfektion dieser Zelllinie wurde etabliert und erreichte eine Effizienz von ungefähr 60
%. Die posttranslationale Regulation der α- und γ-Untereinheiten, insbesondere die
proteolytische Aktivierung funktionierten wie in der Literatur beschrieben, jedoch waren
die Expressionslevel zu gering für weitere Versuche. In der Lunge werden ENaCs
überwiegend in epithelialen Zellen exprimiert. Diese Zellen konnten bisher jedoch nicht
effizient transfiziert werden, ohne Viren einzusetzen. In der vorliegenden Arbeit wurde
jedoch eine effiziente Methode zur Transfektion von primären epithelialen Zellen der Ratte
erarbeitet. Die Expression von transfizierten ENaC-Untereinheiten war in diesen Zellen
deutlich erhöht, weswegen die Etablierung dieses Systems ausschlaggebend für weitere
Versuche ist.
Zusammenfassung
82
Die vorliegende Arbeit beschreibt daher zum ersten Mal die nicht-virale, effiziente
Transfektion von primären alveolaren Zellen und liefert damit ein bedeutendes neues
Werkzeug für die Lungenforschung.
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Appendix
94
Figure Index
Figure 1 Scheme of alveolar Na+-transport ___________________________________________________ 7
Figure 2 Proposed mechanism of CO2 regulated endocytosis of ENaC ____________________________ 14
Figure 3 Scheme of the custom build Ussing chamber._________________________________________ 20
Figure 4 Amiloride-sensitive current during pH variations ______________________________________ 42
Figure 5 Apical application of hypercapnic solution ___________________________________________ 43
Figure 6 Basolateral application of hypercapnic solution _______________________________________ 43
Figure 7 Principle of apical and basolateral membrane permeabilization ___________________________ 44
Figure 8 Ouabain-sensitive Na+-transport during short-term hypercapnia. __________________________ 45
Figure 9 Amiloride-sensitive Na+-transport during short-term hypercapnia _________________________ 46
Figure 10 Effect of 24 h CO2 on Na+-conductance ____________________________________________ 47
Figure 11 Transcription levels of ENaC subunits after 6 h CO2 __________________________________ 48
Figure 12 Transcription levels of ENaC subunits after 24 h CO2 _________________________________ 49
Figure 13 β-subunit of the human epithelial Na+-channel was cloned from human lung tissue __________ 50
Figure 14 Short-term effect of CO2 on β-ENaC-V5 ___________________________________________ 51
Figure 15 Long-term effect of CO2 on β-ENaC-V5____________________________________________ 52
Figure 16 Overview about all ENaC-plasmids that were generated as part of this study _______________ 53
Figure 17 Expression pattern of α-ENaC before and after site-directed mutagenesis __________________ 54
Figure 18 ENaC localisation in soluble fraction and in lipid rafts in A549 cells. _____________________ 55
Figure 19 Stability of cell surface α-ENaC-YFP transfected in A549 cells _________________________ 56
Figure 20 Stability of surface α-ENaC during normocapnia and hypercapnia _______________________ 57
Figure 21 Nedd4-2 dependent cell surface expression of α-ENaC-YFP ____________________________ 58
Figure 22 CO2 dependent stability of α-ENaC-YFP during silencing of Nedd4-2. ____________________ 59
Figure 23 GFP expression of transfected H441 cells___________________________________________ 60
Figure 24 Transfection efficiency of H441 cells achieved by nucleofection_________________________ 60
Figure 25 Viability of H441 cells after Nucleofection _________________________________________ 61
Figure 26 Cotransfection of α β and γ-ENaC in H441 cells _____________________________________ 61
Figure 27 Scheme of the commercially available eGFP-tagged α and γ-ENaC constructs (Origene) used in
this study ____________________________________________________________________________ 62
Figure 28 Expression pattern of α and γ-ENaC in the cell-surface of H441 cells _____________________ 63
Figure 29 Nucleofection of primary rat alveolar epithelial type II cells. ____________________________ 65
Figure 30 Transfection efficiency of ATII cells ______________________________________________ 65
Figure 31 Evaluation of early cytotoxicity in transfected ATII cells ______________________________ 66
Figure 32 Cell number 48 h after Nucleofection ______________________________________________ 67
Figure 33 Viability of transfected ATII cells. ________________________________________________ 67
Figure 34 Electrical resistance 48 h after Nucleofection ________________________________________ 68
Figure 35 Pilot experiment for transfection of ENaC subunits in ATII cells ________________________ 69
Appendix
95
Tables
Table 1 Composition cDNA synthesis (20 µl reaction)_________________________________________ 21
Table 2 Composition real-time PCR (25 µl reaction) __________________________________________ 21
Table 3 Real time PCR conditions ________________________________________________________ 21
Table 4 Real-time PCR Primer ___________________________________________________________ 22
Table 5 Cloning primers and genetic modifications ___________________________________________ 23
Table 6 PCR reaction β-ENaC (25 µl)______________________________________________________ 24
Table 7 PCR conditions for cloning of β-ENaC ______________________________________________ 24
Table 8 Restrictiondigestion for b-ENaC ___________________________________________________ 25
Table 9 Ligation reaction________________________________________________________________ 25
Table 10 PCR reaction for α-ENaC (25 µl) __________________________________________________ 26
Table 11 First PCR condition for cloning of α -ENaC _________________________________________ 26
Table 12 Second PCR condition for cloning of α -ENaC _______________________________________ 26
Table 13 Restriction digestion α-ENaC (40 µl volume) ________________________________________ 27
Table 14 Ligation for α-ENaC T4 ligase kit (20 µl) ___________________________________________ 27
Table 15 Primers for site directed mutagenesis _______________________________________________ 28
Table 16 QuikChange site directed mutagenesis reaction _______________________________________ 28
Table 17 QuikChange site directed mutagenesis thermal profile _________________________________ 28
Table 18 PCR reaction colony-PCR (20 µl) _________________________________________________ 30
Table 19 Antibodies for Western-Blotting __________________________________________________ 31
Table 20 Electronic devices _____________________________________________________________ 35
Table 21 Reagents _____________________________________________________________________ 36
Table 22 Buffers ______________________________________________________________________ 39
Appendix
96
8. Eidesstattliche Versicherung
„Ich erkläre: Ich habe die vorgelegte Dissertation selbständig und ohne unerlaubte fremde
Hilfe und nur mit den Hilfen angefertigt, die ich in der Dissertation angegeben habe. Alle
Textstellen, die wörtlich oder sinngemäß aus veröffentlichten Schriften entnommen sind,
und alle Angaben, die auf mündlichen Auskünften beruhen, sind als solche kenntlich
gemacht.
Bei den von mir durchgeführten und in der Dissertation erwähnten Untersuchungen habe
ich die Grundsätze guter wissenschaftlicher Praxis, wie sie in der „Satzung der JustusLiebig-Universität Gießen zur Sicherung guter wissenschaftlicher Praxis“ niedergelegt
sind, eingehalten.“
Benno Buchbinder
Appendix
97
9. Danksagung
Während der Durchführung dieser Arbeit haben mich viele Menschen begleitet, unterstützt
und gefördert und bei diesen möchte ich mich an dieser Stelle herzlich bedanken:
Meinem Betreuer Dr. István Vadász möchte ich danken für die Bereitstellung des Themas,
für die Freiheiten während der Planung und Durchführung der Experimente, sowie für die
Unterstützung und Kameradschaft, die ich in dieser Zeit bekommen habe.
Ich danke Prof. Werner Seeger für die Aufnahme ins Graduiertenkolleg „MBML“, die mir
nicht nur die Durchführung dieser Arbeit, sondern auch umfassendes wissenschaftliches
Training und die rege Teilnahme an internationalen Kongressen ermöglichte. Des Weiteren
danke ich ihm für die Betreuung dieser Arbeit, die ich dadurch extern am Fachbereich
Medizin durchführen durfte.
Prof. Wolfgang Clauss begleitete mich bereits während meiner Diplomarbeit. Ihm danke
ich für die Betreuung im Fachbereich Biologie.
Ein besonderer Dank geht an Ingrid Breitenborn-Müller und Andrea Mohr für ihre
bedingungslose Unterstützung im Laboralltag. Ohne sie wäre diese Arbeit nicht möglich
gewesen.
Bei meinen Kollegen möchte ich mich ebenfalls bedanken, insbesondere Miriam
Wessendorf, Yasmin Buchäckert und Nieves Gabrielli, die mich über weite Strecken in
den letzten Jahren begleitet haben.
Ganz besonders möchte ich meiner Frau Anja danken, die unendlich viel Geduld bewiesen
hat und mich trotz der Entbehrungen oder gerade wegen ihnen bestärkt hat diese Arbeit
abzuschließen.
Zuletzt möchte ich mich für die fortwährende Unterstützung meiner Familie bedanken, die
mir ein finanziell absolut sorgenfreies Studium ermöglichte. Vielen Dank auch für eure
Anteilnahme am Gelingen dieser Arbeit.
Der Lebenslauf wurde aus der elektronischen
Version der Arbeit entfernt.
The curriculum vitae was removed from the
electronic version of the paper.
Appendix
99
Orginale Publikationen
BA Grzesik, CU Vohwinkel, RE Morty, K Mayer, S Herold, W Seeger, I Vadász „Efficient gene
delivery to primary alveolar epithelial cells by nucleofection” Am J Physiol Lung Cell Mol
Physiol. [in press]
K Richter, KP Kiefer, BA Grzesik, WG Clauss, M Fronius „Hydrostatic pressure activates ATPsensitive K+ channels in lung epithelium by ATP-release through pannexin/connexin
hemichannels FASEB Journal [in press]
I Vadász, LA Dada, A Briva, IT Helenius, K Sharabi, LC Welch, AM Kelly, BA Grzesik, GR
Budinger, J Liu, W Seeger, GJ Beitel, Y Gruenbaum, JI Sznajder. „Evolutionary conserved role of
c-Jun-N-terminal kinase in CO2-induced epithelial dysfunction.” PLoS One. 2012;7(10):e46696
Y Buchaeckert, S Rummel, CU Vohwinkel, NM Gabrielli, BA Grzesik, K Mayer, S Herold, RE
Morty, W Seeger, I Vadász. „Megalin mediates transepithelial albumin clearance from the
alveolar space of intact rabbit lungs.” J Physiol. 2012 Oct 15;590(Pt 20):5167-81
Vorträge und Poster
BA Grzesik, R Ruhl, N Gabrielli, W Seeger, W Kummer, U Pfeil, I Vadasz.
„IntermedinEnhances Alveolar Edema Clearance By Promoting Na,K-ATPase Exocytosis In A
Protein Kinase A-Dependent But cAMP-Independent Manner“ Am J Respir Crit Care Med 185;
2012: A3521
BA Grzesik, NM Gabrielli, M Fronius, RE Morty, W Seeger, JI Sznajder, I Vadasz. “Long-Term
Hypercapnia Impairs ENaC Function In Alveolar Epithelial Monolayers: A Role For
Ubiquitination?” Am J Respir Crit Care Med 183; 2011: A4228
NM Gabrielli, BA Grzesik, LA Dada, W Seeger, JI Sznajder, I Vadasz. „Effects Of Hypercapnia
On Na,K-ATPase Stability And Epithelial Cell Adhesion In Human Alveolar Epithelial Cells”
Am J Respir Crit Care Med 183; 2011: A4232
BA Grzesik, M Fronius, W Seeger, RE Morty, JI Sznajder, I Vadasz. „Short-term Hypercapnia
Differentially Regulates Na,K-ATPase And ENaC Function In Polarized H441 Cell
Monolayers” Am. J. Respir. Crit. Care Med. 2010; 181: A6462.
Appendix
100
K Richter, BA Grzesik, R Bogdan, W Clauss, M Fronius. „ATP-sensitive K+-channels In
Pulmonary Epithelium (Xenopus laevis) Are Activated By Mechanical Stress.“ Acta Physiologica
2010; Volume 198, Supplement 677:P-TUE-42
I Vadasz, LA Dada, A Briva, HE Trejo, LC Welch, AM Kelly, K Sharabi, BA Grzesik, J Liu, Y
Gruenbaum, W Seeger, JI Sznajder. „Central Role Of C-Jun N-terminal Kinase In Hypercapniainduced Alveolar Epithelial Dysfunction” Am J Respir Crit Care Med 181;2010:A6459
C Veith, BA Grzesik, R Bogdan, WG Clauss, M Fronius. “Mechanical Stress Stimulates ATP
Release And Activates KATP-channels In Xenopus Lung Epithelium” Acta Physiologica 2008;
Volume 192, P140

Impact of hypercapnia on alveolar Na+

Transcription

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